Characteristics of common pathogens of urogenital tract among outpatients in Shanghai, China from 2016 to 2021.

Background: Ureaplasma urealyticum, Chlamydia trachomatis, and Neisseria gonorrhoeae are the prevalent causes of several genital diseases worldwide; however, their characteristics in different genders have not been well documented in Shanghai. The aim of this study is to describe the prevalence of common pathogens among outpatients, considering variations by gender and age. Methods: From January 1, 2016, to December 31, 2021, the urogenital swabs of 16216 outpatients aged 3-95 years from two general hospitals in Shanghai were collected. All participants' swabs were investigated for U. urealyticum, C. trachomatis, and N. gonorrhoeae by isothermal RNA-based simultaneous amplification and testing. The basic information of all participants was also recorded, including age and gender. The chi-square test was used to compare the prevalence between different genders, age groups, and infection patterns. Results: There were 5,744 patients (35.42%) with positive samples whose ages ranged from 7 to 80 years (33.23 ± 8.63 years), and 62.14% of them were women. The most common pathogen detected was U. urealyticum (85.08%). The highest prevalence rate of all three pathogens was found in patients aged ≤ 20 years (40.53%, 95% confidence intervals [CI]: 33.80%-47.63%). The prevalent rate of U. urealyticum was higher in men (33.36%, 95% CI: 32.19%-34.55%). The overall prevalence rates of U. urealyticum, C. trachomatis, and N. gonorrhoeae were 30.14% (95% CI: 29.44%-30.85%), 6.00% (95% CI: 5.64%-6.38%), and 2.10% (95% CI: 1.89%-2.33%). Conclusions: Ureaplasma urealyticum was the most prevalent pathogen in the population, and its prevalence decreased with age. Young men aged ≤ 20 years were more frequently infected. Regular screening for sexually transmitted pathogens in different genders and age groups are warranted, particularly in young men.

A rapid and high-throughput multiplex genetic detection assay for detection, semi-quantification and virulence genotyping of Helicobacter pylori in non-invasive oral samples.

Aim: This study established a high-throughput multiplex genetic detection assay (HMGA) for rapid identification, semi-quantification and virulence analysis of Helicobacter pylori directly from the clinical non-invasive oral samples. Methods: The gastric mucosa and oral samples were collected from 242 patients in Shanghai from 2021 to 2022. All the samples were detected by routine clinical tests for H. pylori and Sanger sequenced for inconsistent results. A new multiplex PCR assay providing results within 4 hours was designed and optimized involving fluorescent dye-labeled specific primers targeted 16S rRNA gene, semi-quantitative gene ureC and 10 virulence genes of H. pylori. Semi-quantification was carried out by simulating the serial 10-fold dilutions of positive oral samples, and the H. pylori loads in different clinical samples were further compared. The mixed plasmids of virulence genes vacA s1, vacA m1 and vacA m2 were used to evaluate the performance on different genotypes. The consistency of 10 virulence genes in gastric mucosa, saliva, mouthwash and dental plaque of H. pylori-positive patients was compared. Results: The non-invasive HMGA was highly specific for detection of all 12 targets of H. pylori and human internal reference gene ß-globin, and the sensitivity to all target genes could reach 10 copies/µL. Compared with routine clinical tests and sequencing, non-invasive HMGA has a high level (>0.98) of sensitivity, specificity, accuracy, PPV, NPV and kappa coefficient for direct detection of H. pylori in oral samples. Moreover, by detecting peak area levels of ureC, it was confirmed that the H. pylori loads in gastric mucosa were significantly higher than those of the three kinds of oral samples (p<0.05). We also found that 45.0% (91/202) of patients had different H. pylori virulence genes in different oral samples. The concordance of positive detection rates of each virulence gene between saliva and gastric mucosa was more than 78% (p<0.05). Conclusion: The non-invasive HMGA proved to be a reliable method for the rapid H. pylori identification, semi-quantification and detection of 10 virulence genes directly in oral samples, providing a new idea for non-invasive detection of H. pylori.

Identification of Intestinal Microbial Community in Gallstone Patients with Metagenomic Next-Generation Sequencing.

Gallstone disease (GD) is one of the most common gastrointestinal diseases worldwide. Nowadays, intestinal microbiota are thought to play important roles in the formation of gallstones. In our study, human fecal samples were extracted for metagenomic next-generation sequencing (mNGS) on the Illumina HiSeq platform, followed by bioinformatics analyses. Our results showed that there was a particular intestinal micro-ecosystem in GD patients. In contrast to healthy people, the sequences of Bacteroidetes, Bacteroides and Thetaiotaomicron were obviously more abundant in GD patients at phylum, genus and species levels, respectively. On the other hand, the glycan metabolism and drug resistance, especially for the ß-lactams, were the most profound functions of gut microbes in GD patients compared to those in normal subjects. Furthermore, a correlation analysis drew out that there existed a significant relationship between the serum levels of biochemical indicators and abundances of intestinal microbes in GD patients. Our results illuminate both the composition and functions of intestinal microbiota in GD patients. All in all, our study can broaden the insight into the potential mechanism of how gut microbes affect the progression of gallstones to some extent, which may provide potential targets for the prevention, diagnosis or treatment of GD.

A New Multiplex Genetic Detection Assay Method for the Rapid Semi-Quantitative Detection of Six Common Curable Sexually Transmitted Pathogens From the Genital Tract.

Background: Sexually transmitted infections (STIs) are some of the most common communicable conditions and exert impact on the health and lives of many hundreds of millions of people across the world every year. Screening high-risk populations and conducting comprehensive detection tests would lead to a significant improvement in preventing the transmission of STIs and help us to provide rapid treatment to those affected. Here, we successfully established and validated a novel high-throughput multiplex gene detection system (HMGS) for the simultaneous and semiquantitative detection of six important curable sexually transmitted pathogens in a single reaction from secretions samples. Method: Fluorescently labeled primers were designed to target specific conserved and single-copy gene fragments of Ureaplasma urealyticum (U. urealyticum), Mycoplasma hominis (M. hominis), Chlamydia trachomatis (C. trachomatis), Neisseria gonorrhoeae (N. gonorrhoeae), Trichomonas vaginalis (T. vaginalis), and Treponema pallidum (T. pallidum). The specificity and sensitivity of the STI-HMGS was validated and optimized using plasmids and quantitative genomic DNA. Next, we validated the performances of the STI-HMGS for clinical application by testing samples of clinical secretions collected from patients who visited the gynecology and urology outpatient clinics of our reproductive medicine center. Results derived from the STI-HMGS were then compared with three approved commercialized kits that used to detect U. urealyticum, C. trachomatis and N. gonorrhoeae, respectively, followed by further validation with Sanger sequencing for all pathogens. Finally, a comprehensive analysis of epidemiology was performed among different subgroups to investigate the association between infection rates and clinically-relevant information. Results: The sensitivity of STI-HMGS for six target genes was 10 copies/µL. Data derived from the detection of 381 clinical secretions demonstrated that the STI-HMGS exhibited high concordance rate compared with approved commercialized kits and almost 100% sensitivity and specificity for the detection of six sexually transmitted pathogens when validated by Sanger sequencing. Semi-quantitative analysis found that STIs caused by N. gonorrhoeae had a significantly higher (P<0.05) pathogen load than the other pathogens. Infections caused by C. trachomatis were significantly more common in younger individuals (P<0.05). We also found that U. urealyticum infections were more likely to happen in females; while the males were more affected by N. gonorrhoeae (P<0.05). Conclusions: STI-HMGS proved to be an efficient method for the semi-quantitative detection of six important curable sexually transmitted pathogens and therefore represents an alternative method for the clinical detection and monitoring of STIs.

Can Chemical Analysis Predict Wine Aging Capacity?

Oxidation is the limiting factor in wine aging, and recently some famous wines have exhibited unexpected premature oxidation. Antioxidant assays may provide a means to assess a wine's aging potential by measuring its capacity to chemically reduce reagent components. Correlations between antioxidant activity and wine components have the highest value with flavanols, notable for their catechol and phloroglucinol moieties. Both FRAP and DPPH based methods respond strongly to catechol groups, but these functional groups do not protect wine from oxidation. An ideal assay for wine aging capacity would respond selectively to thiols, phloroglucinol moieties, SO2 and other antioxidants capable of reducing quinones. A definitive test will be to compare the various assays against the shelf life of a number of commercial wines.

The composition of intestinal microbiota and its association with functional constipation of the elderly patients.

Aim: To identify intestinal microbiota compositions in elderly functional constipation (FC) patients. Materials & methods: Fecal samples from 61 FC patients and 48 healthy age-matched volunteers were analyzed through 16S rRNA gene sequencing. Results: The intestinal microbiota compositions of FC patients were significantly different from healthy controls. Additionally, the species diversity of healthy controls was greater than that of FC patients. Indeed, the abundance of Firmicutes and Proteobacteria was significantly decreased, whereas that of Bacteroides, Prevotella, Lactococcus, Ruminococcus and Butyricimonas was remarkably increased in FC patients. Conclusion: Elderly FC patients appear to have a unique intestinal microbiota profile. Our findings should provide insight regarding the pathogenic mechanism of FC and evidence for exploring new therapeutic strategies in elderly FC patients.

Clinical application of a multiplex genetic pathogen detection system remaps the aetiology of diarrhoeal infections in Shanghai.

BACKGROUND: Culture-based diagnostic methods cannot achieve rapid and precise diagnoses for the identification of multiple diarrhoeal pathogens (DPs). A high-throughput multiplex genetic detection system (HMGS) was adapted and evaluated for the simultaneous identification and differentiation of infectious DPs and a broad analysis of DP infection aetiology. RESULTS: DP-HMGS was highly sensitive and specific for DP detection compared with culture-based techniques and was similar to singleplex real-time PCR. The uniform level of sensitivity of DP-HMGS for all DPs allowed us to remap the aetiology of acute diarrhoeal infections in Shanghai, correcting incidences of massively underdiagnosed DP species with accuracy approaching that of sequencing-based methods. The most frequent DPs were enteropathogenic Escherichia coli, rotavirus and Campylobacter jejuni. DP-HMGS detected two additional causes of infectious diarrhoea that were previously missed by routine culture-based methods: enterohemorrhagic E. coli and Yersinia enterocolitica. We demonstrated the age dependence of specific DP distributions, especially the distributions of rotavirus, intestinal adenovirus and Clostridium difficile in paediatric patients as well as those of dominant bacterial infections in adults, with a distinct "top 3" pattern for each age group. Finally, the multiplexing capability and high sensitivity of DP-HMGS allowed the detection of infections co-induced by multiple pathogens (approximately 1/3 of the cases), with some DPs preferentially co-occurring as infectious agents. CONCLUSIONS: DP-HMGS has been shown to be a rapid, specific, sensitive and appropriate method for the simultaneous screening/detection of polymicrobial DP infections in faecal specimens. Widespread use of DP-HMGS is likely to advance routine diagnostic and clinical studies on the aetiology of acute diarrhoea.

Validation of a High-Throughput Multiplex Genetic Detection System for Helicobacter pylori Identification, Quantification, Virulence, and Resistance Analysis.

Helicobacter pylori (H. pylori) infection is closely related to various gastroduodenal diseases. Virulence factors and bacterial load of H. pylori are associated with clinical outcomes, and drug-resistance severely impacts the clinical efficacy of eradication treatment. Existing detection methods are low-throughput, time-consuming and labor intensive. Therefore, a rapid and high-throughput method is needed for clinical diagnosis, treatment, and monitoring for H. pylori. High-throughput Multiplex Genetic Detection System (HMGS) assay was established to simultaneously detect and analyze a set of genes for H. pylori identification, quantification, virulence, and drug resistance by optimizing the singlet-PCR and multiple primers assay. Twenty-one pairs of chimeric primers consisted of conserved and specific gene sequences of H. pylori tagged with universal sequence at the 5' end were designed. Singlet-PCR assay and multiple primers assay were developed to optimize the HMGS. The specificity of HMGS assay was evaluated using standard H. pylori strains and bacterial controls. Six clinical isolates with known genetic background of target genes were detected to assess the accuracy of HMGS assay. Artificial mixed pathogen DNA templates were used to evaluate the ability to distinguish mixed infections using HMGS assay. Furthermore, gastric biopsy specimens with corresponding isolated strains were used to assess the capability of HMGS assay in detecting biopsy specimens directly. HMGS assay was specific for H. pylori identification. HMGS assay for H. pylori target genes detection were completely consistent with the corresponding genetic background. Mixed infection with different drug-resistant isolates of H. pylori could be distinguished by HMGS assay. HMGS assay could efficiently diagnose H. pylori infection in gastric biopsy specimens directly. HMGS assay is a rapid and high throughput method for the simultaneous identification and quantification of H. pylori, analysis of virulence and drug resistance in both isolated strains and biopsy specimens. It could also be used to distinguish the mixed infection with different resistant genotype strains. Furthermore, HMGS could detect H. pylori infection in gastric biopsy specimens directly.

Direct detection of Helicobacter pylori in biopsy specimens using a high-throughput multiple genetic detection system.

AIM: We evaluated the direct high-throughput multiple genetic detection system (dHMGS) for Helicobacter pylori in gastric biopsies. MATERIALS & METHODS: One hundred and thirty-three specimens were concurrently analyzed by dHMGS, rapid urease test, culture and sequencing. RESULTS: dHMGS was highly sensitive and specific for H. pylori identification compared with culture and rapid urease test. The correlation coefficient of the quantitative standard curve was R2 = 0.983. A significant difference in the relative H. pylori DNA abundance was found in different gastroduodenal diseases. Concordance rates between dHMGS and sequencing for resistance mutations were 97.1, 100.0, 85.3 and 97.1%, respectively. Finally, dHMGS could efficiently distinguish mixed infection in biopsy specimens. CONCLUSION: The dHMGS could efficiently diagnose and quantify H. pylori burden in biopsies, simultaneously screening for virulence, antibiotic resistance and presence of the multistrain infections.

A high-throughput multiplex genetic detection system for Helicobacter pylori identification, virulence and resistance analysis.

AIM: We established a high-throughput multiplex genetic detection system (HMGS) for identification of Helicobacter pylori with concomitant analysis of virulence and drug resistance. MATERIALS & METHODS: Confirmed 132 H. pylori cultures from gastric biopsies were screened by 20-gene site-HMGS, sequencing and E-test. RESULTS: HMGS was highly sensitive and specific for H. pylori identification. Concordance rate between HMGS and sequencing averaged 94.5% (virulence genes) and 97.3% (resistance genes). Observed resistance rates to four mainstream antibiotics were high, except for amoxicillin. Significant association between virulence genotype and risks for specific gastrointestinal diseases was found for five genes. Metronidazole resistance in peptic ulcer patients was significantly higher. CONCLUSION: HMGS is an effective method for H. pylori identification and analysis of virulence and drug resistance.

A Creative Helicobacter pylori Diagnosis Scheme Based on Multiple Genetic Analysis System: Qualification and Quantitation.

BACKGROUND: Currently, several diagnostic assays for Helicobacter pylori (H. pylori) are available, but each has some limitations. Further, a high-flux quantitative assay is required to assist clinical diagnosis and monitor the effectiveness of therapy and novel vaccine candidates. METHODS: Three hundred and eighty-seven adult patients [nonulcer dyspepsia (NUD) 295, peptic ulcer disease (PUD) 77, gastric cancer (GC) 15] were enrolled for gastrointestinal endoscopies. Three biopsy samples from gastric antrum were collected for the following tests: culture, rapid urease test (RUT), histopathology, conventional polymerase chain reaction (PCR), and Multiple Genetic Analysis System (MGAS). The diagnostic capability of H. pylori for all methods was evaluated through the receiver operating characteristic (ROC) curves. RESULTS: Based on the gold standard, the sensitivity and specificity of MGAS were 92.9 and 92.4%, and positive predict value (PPV) and negative predict value (NPV) were 96.0 and 87.1%, respectively. All the above parameters of MGAS were higher than that of culture (except its specificity), RUT and histopathology, and nearly closed to that of conventional PCR. The area under curve (AUC) was 0.7575 (Culture), 0.8870 (RUT), 0.9000 (Histopathology), 0.9496 (Conventional PCR), and 0.9277 (MGAS). No significant statistical difference was observed for the H. pylori DNA load in different disease groups (p = .067). In contrast, a statistically significant difference in the H. pylori DNA copy number was observed based on age (p = .043) and gender (p = .021). CONCLUSIONS: The data showed that MGAS performed well in detecting H. pylori infection. Furthermore, the quantitative analysis showed that the load of H. pylori was significantly different within both age and gender groups. These results suggested that MGAS could be a potential alternative method for clinical detection and monitoring of the effectiveness of H. pylori therapy.

[Effect of osteoprotegerin gene polymorphism on bone mass in postmenopausal women].

OBJECTIVE: To investigate the effect of promoter region polymorphism of osteoprotegerin (OPG) gene on bone mineral density (BMD) and markers of bone turnover in postmenopausal women. METHODS: The OPG genotype were determined by PCR-RFLP in 259 postmenopausal women of Han race in Shanghai area, and BMD at lumbar-spine, femoral neck, trochanter and Ward's triangle etc., were measured by dual-energy X-ray absorptiometry (DEXA). Some markers of bone turnover were also detected. RESULTS: Hardy-Weinberg equilibrium was evident for OPG polymorphism. The frequency of the genotype is as following: TT59%, TC28%, CC13%. Postmenopausal women were divided into two groups. Women aged less than 65 years old were in group I, and aged 65 years old and over were in group II. In group I the BMD at femur neck area was 0.64 g/cm(2) +/- 0.11 g/cm(2), 0.67 g/cm(2) +/- 0.11 g/cm(2), 0.68 g/cm(2) +/- 0.14 g/cm(2) of TT, TC, CC genotypes respectively, among which a significant difference was observed (P < 0.05 by ANOVA), and the significant difference also presented after adjusted by age and BMI, but not found the significant difference in other markers. In group II, significant link was not observed between OPG polymorphism and BMD with markers of bone turnover. CONCLUSION: OPG polymorphism may have an effect on the BMD of postmenopausal women aged less than 65. It is possibly that the role of OPG gene mainly on the obtaining of peak BMD in women.