Typing of Neisseria Gonorrhoeae isolates in Shenzhen, China from 2014-2018 reveals the shift of genotypes associated with antimicrobial resistance.

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-STARST497, MLSTST7365, and MLSTST7360 were typically associated with decreased susceptibility to ceftriaxone. Furthermore, the internationally spreading ESC-resistant clone MLSTST1901 has been prevalent at least in 2014 in Shenzhen and showed a significant increase during 2014-2018. Additionally, MLSTST7363 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.

Multiplex PCR and Nanopore Sequencing of Genes Associated with Antimicrobial Resistance in Neisseria gonorrhoeae Directly from Clinical Samples.

BACKGROUND: Antimicrobial resistance (AMR) of Neisseria gonorrhoeae has spread worldwide. Rapid and comprehensive methods are needed to describe N. gonorrhoeae AMR profiles accurately. A method based on multiplex amplicon sequencing was developed to simultaneously sequence 13 genes related to AMR in N. gonorrhoeae directly from clinical samples. METHODS: Nine N. gonorrhoeae strains were used for the establishment and validation of the method. Eleven urethral swabs and their corresponding cultured isolates were matched as pairs to determine the accuracy of the method. Mock samples with different dilutions were prepared to determine the sensitivity of the method. Five nongonococcal Neisseria strains and 24 N. gonorrhoeae negative clinical samples were used to evaluate the cross-reactivity. Finally, the method was applied to 64 clinical samples to assess its performance. RESULTS: Using Sanger sequencing as a reference method, sequences recovered from amplicon sequencing had a base accuracy of over 99.5% and the AMR sites were correctly identified. The limit of detection (LOD) was lower than 31 copies/reaction. No significant cross-reactivity was observed. Furthermore, target genes were successfully recovered from 64 clinical samples including 9 urines, demonstrating this method could be used in different types of samples. For clinical samples, the results can be obtained within a time frame of 7 h 40 min to 10 h 40 min, while for isolates, the turnaround time was approximately 2 h shorter. CONCLUSIONS: This method can serve as a versatile and convenient culture-free diagnostic method with the advantages of high sensitivity and accuracy.

A molecular screening assay to identify Chlamydia trachomatis and distinguish new variants of C. trachomatis from wild-type.

Chlamydia trachomatis is the most common sexually transmitted pathogen globally, causing serious health problems and representing a burden on public health. A new variant of C. trachomatis (nvCT) that carries mutations (C1514T, C1515T and G1523A) in the 23S rRNA gene has eluded detection in Aptima Combo 2 assays. This has led to false negatives in diagnostics tests and poses a challenge for C. trachomatis diagnostics on a global level. In this study, we developed a simple and cost-effective assay to identify C. trachomatis, with a potential application to screen for nvCT. We developed a screening assay based on high-resolution melting (HRM), targeting the 23S rRNA gene and cryptic plasmid. To evaluate the performance of the assay, 404 archived C. trachomatis DNA specimens and 570 extracted clinical specimens were analysed. Our HRM assay not only identified C. trachomatis in clinical specimens, but also correctly differentiated nvCT carrying C1514T, C1515T and G1523A mutations from the wild-type. We observed no cross-reactions with other clinically related agents, and the limit of detection was 11.26 (95% CI; 7.61-31.82) copies per reaction. Implementation of this screening assay could reduce detection times and costs for C. trachomatis diagnoses, and facilitate increased research on the presence and monitoring of nvCT.

Surveillance and molecular epidemiology of Neisseria gonorrhoeae isolates in Shenzhen, China, 2010-2017.

OBJECTIVES: The development and emergence of antimicrobial resistance in Neisseria gonorrhoeae (NG) have become a major public-health problem worldwide. This study aimed to analyse the antimicrobial susceptibility and molecular characteristics of NG isolates in Shenzhen, China. METHODS: A total of 1282 NG isolates were consecutively collected between 2010 and 2017. Patient demographic information was also collected. MICs of ceftriaxone, spectinomycin, ciprofloxacin, azithromycin and penicillin were determined by agar dilution. Isolates were genotyped using N. gonorrhoeae multi-antigen sequence typing (NG-MAST). RESULTS: Among the isolates, 97.4% were resistant to ciprofloxacin and 68.2% to penicillin. Moreover, 5.0% showed decreased susceptibility to ceftriaxone (CROD) and 17.3% were resistant to azithromycin (AZM-R); 1.3% were simultaneously CROD and AZM-R. All isolates were susceptible to spectinomycin. Increasing ceftriaxone MICs were found from 2010 to 2017. A total of 427 sequence types (STs) and 68 genogroups were identified from 724 isolates. ST5061, ST3741 and ST1766 were observed across the study years. ST14638 (n = 3) was predominant among 32 CROD isolates. Prevalent STs were ST5061 (n = 6), ST1866 (n = 5) and ST11133 (n = 5) among 96 AZM-R isolates. CONCLUSIONS: A high prevalence of isolates resistant to ciprofloxacin and penicillin was found in this study. Azithromycin, one antimicrobial of dual antimicrobial therapy recommended by the WHO, showed a high prevalence of resistance. The other, ceftriaxone, can be used continuously in this region owing to lower resistance levels. However, the emergence of CROD and decreasing susceptibility to ceftriaxone indicate that continuous antimicrobial resistance surveillance is essential.

Multiplex High-Resolution Melting Assay for Simultaneous Identification of Molecular Markers Associated with Extended-Spectrum Cephalosporins and Azithromycin Resistance in Neisseria gonorrhoeae.

Antimicrobial resistance in Neisseria gonorrhoeae persists as a major public health concern globally. We developed and evaluated a multiplex assay that relied on high-resolution melting (HRM) technology as a rapid, simple, and cost-effective method for simultaneously detecting and identifying different molecular markers associated with extended-spectrum cephalosporins (ESCs) and azithromycin (AZM) resistance in N. gonorrhoeae. Forty-eight well-characterized N. gonorrhoeae clinical isolates were selected for initial assay establishment. The multiplex HRM assays were able to accurately identify different nucleotide variations of the antimicrobial resistance determinants related to ESCs and AZM resistance. Specificity and cross-reactivity were assessed by testing 15 nongonococcal strains. Then, the assay was validated on 218 archived DNA specimens that had been sequenced using whole-genome sequencing technology. Compared with whole-genome sequencing, these assays had a sensitivity of 98.6%, with a specificity of 99.2%. For further validation of the assay's performance, a total of 338 samples (156 clinical isolates and 182 clinical specimens) were screened using the multiplex HRM assay. The results showed good concordance with the results of PCR sequencing. Given its rapidity (within 90 minutes), ease of performing, and low cost (<$1.00 per sample), this method may be applied to large-scale epidemiologic programs for increasing surveillance of ESCs and AZM resistance in N. gonorrhoeae.

Determining antimicrobial resistance profiles and identifying novel mutations of Neisseria gonorrhoeae genomes obtained by multiplexed MinION sequencing.

Gonorrhea is one of the most common sexually transmitted diseases worldwide. To cure infection and prevent transmission, timely and appropriate antimicrobial therapy is necessary. Unfortunately, Neisseria gonorrhoeae, the etiological agent of gonorrhea, has acquired nearly all known mechanisms of antimicrobial resistance (AMR), thereby compromising the efficacy of antimicrobial therapy. Treatment failure resulting from AMR has become a global public health concern. Whole-genome sequencing is an effective method to determine the AMR characteristics of N. gonorrhoeae. Compared with next-generation sequencing, the MinION sequencer (Oxford Nanopore Technologies (ONT)) has the advantages of long read length and portability. Based on a pilot study using MinION to sequence the genome of N. gonorrhoeae, we optimized the workflow of sequencing and data analysis in the current study. Here we sequenced nine isolates within one flow cell using a multiplexed sequencing strategy. After hybrid assembly with Illumina reads, nine integral circular chromosomes were obtained. By using the online tool Pathogenwatch and a BLAST-based workflow, we acquired complete AMR profiles related to seven classes of antibiotics. We also evaluated the performance of ONT-only assemblies. Most AMR determinants identified by ONT-only assemblies were the same as those identified by hybrid assemblies. Moreover, one of the nine assemblies indicated a potentially novel antimicrobial-related mutation located in mtrR which results in a frame-shift, premature stop codon, and truncated peptide. In addition, this is the first study using the MinION sequencer to obtain complete genome sequences of N. gonorrhoeae strains which are epidemic in China. This study shows that complete genome sequences and antimicrobial characteristics of N. gonorrhoeae can be obtained using the MinION sequencer in a simple and cost-effective manner, with hardly any knowledge of bioinformatics required. More importantly, this strategy provides us with a potential approach to discover new AMR determinants.

Associations of plasma metal concentrations with the decline in kidney function: A longitudinal study of Chinese adults.

Metals are widespread pollutants in the environment which have been reported to be associated with kidney dysfunction in many existing epidemiological studies. However, most of the studies are cross-sectional design and mainly focus on several toxic metals including arsenic, lead and cadmium. Therefore, we conducted this prospective study within the Dongfeng-Tongji cohort to evaluate the associations of plasma multiple metals with the decline in kidney function among Chinese middle-aged and elderly. In total, 1434 participants free of chronic diseases at baseline were included in analysis. We measured baseline plasma concentrations of 23 metals and calculated estimated glomerular filtration rate (eGFR) using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation based on serum creatinine, age, sex and ethnicity. Bonferroni correction was used for multiple testing to reduce the probability of a type I error. Principal component analysis was conducted to evaluate the combined effect of multiple metal co-exposure. Most of the plasma metal concentrations were within the literature reported reference values, whereas the concentration of lead and nickel exceeded the guideline value. We found that plasma concentrations of aluminum, arsenic, barium, lead, molybdenum, rubidium, strontium, vanadium and zinc were significantly associated with the decline in kidney function measured by annual eGFR decline, rapid renal function decline (defined as an annual decline in eGFR ≥ 5 mL/min/1.73 m2) or incident eGFR < 60 mL/min/1.73 m2, with the adjusted beta coefficients (95% CI) for annual eGFR decline 0.50 (0.30, 0.69), 0.98 (0.74, 1.23), 0.56 (0.32, 0.79), 0.21 (0.03, 0.39), 0.35 (0.16, 0.54), 0.94 (0.71, 1.17), 0.37 (0.15, 0.60), 0.78 (0.54, 1.02), and 0.74 (0.57, 0.91), respectively. The metals exposures were linked with increased risks of impaired kidney function. Associations of principal components representing these metals with the decline in kidney function were significant and suggest a possible additional health risk by co-exposure. Participants engaged in manufacturing had higher plasma levels of several metals compared with those who had been involved in management- or administration-related work. Our findings suggest that exposure to multiple metals contribute to the decline in kidney function among the middle-aged and elderly. Co-exposure to multiple metals may have synergetic effect on the kidney function. Further studies are warranted to confirm our findings and clarify the potential mechanisms.

Longer time spent in bed attempting to sleep is associated with rapid renal function decline: the Dongfeng-Tongji cohort study.

INTRODUCTION: Prospective evidence on the relation between time in bed and renal dysfunction remains limited. We aimed to investigate the association of time spent in bed attempting to sleep (TSBS) with renal function decline in a middle-aged and elderly Chinese population. METHODS: About 16,733 eligible participants with a mean age of 62.3 years at baseline were included. Rapid renal function decline was defined as (baseline eGFR - revisit eGFR)/years of follow-up ≥5 mL/min per 1.73 m2/year. A total of 1738 study participants experienced rapid renal function decline after a median 4.6-year follow-up. Logistic regression models were used for multivariate analyses. RESULTS: The adjusted odds ratio (OR) of rapid renal function decline was 1.18 (95% CI: 1.02, 1.37) for TSBS ≥9 h/night compared with TSBS 7 to <8 h/night. This association remained significant (OR = 1.19, 95% CI: 1.03, 1.38) after further adjustment for sleep quality, midday napping and usage of sleeping pills. Particularly, the association appeared to be prominent in individuals with diabetes. CONCLUSIONS: Longer TSBS (≥9 h) was independently associated with an increased risk of rapid renal function decline. Our findings emphasized the importance to have optimal TSBS. Key messages Our study firstly investigated the association between time spent in bed attempting to sleep (TSBS) and renal dysfunction in Chinese adults. Compared with individuals TSBS 7 to <8 h, individuals with TSBS ≥9 h had 19% increased risk for rapid renal function decline after adjustment for multivariate confounders. The association appeared to be prominent in individuals with diabetes.

Plasma metabolomics identified novel metabolites associated with risk of type 2 diabetes in two prospective cohorts of Chinese adults.

BACKGROUND: Metabolomics studies in Caucasians have identified a number of novel metabolites in association with the risk of type 2 diabetes (T2D). However, few prospective metabolomic studies are available in Chinese populations. In the present study, we sought to identify novel metabolites consistently associated with incident T2D in two independent cohorts of Chinese adults. METHODS: We performed targeted metabolomics (52 metabolites) of fasting plasma samples by liquid chromatography-mass spectrometry in two prospective case-control studies nested within the Dongfeng-Tongji (DFTJ) cohort and Jiangsu Non-communicable Disease (JSNCD) cohort. After following for 4.61 ± 0.15 and 7.57 ± 1.13 years, respectively, 1039 and 520 eligible participants developed incident T2D in these two cohorts, and controls were 1:1 matched with cases by age (± 5 years) and sex. Multivariate conditional logistic regression models were constructed to identify metabolites associated with future T2D risk in both cohorts. RESULTS: We identified four metabolites consistently associated with an increased risk of developing T2D in the two cohorts, including alanine, phenylalanine, tyrosine and palmitoylcarnitine. In the meta-analysis of two cohorts, the odds ratios (95% confidence intervals, CIs) comparing extreme quartiles were 1.79 (1.32-2.42) for alanine, 1.91 (1.41-2.60) for phenylalanine, 1.85 (1.37-2.48) for tyrosine and 1.63 (1.21-2.20) for palmitoylcarnitine (all Ptrend ≤ 0.01). CONCLUSIONS: We confirmed the association of alanine, phenylalanine and tyrosine with future T2D risk and further identified palmitoylcarnitine as a novel metabolic marker of incident T2D in two prospective cohorts of Chinese adults. Our findings might provide new aetiological insight into the development of T2D.