Antibiotics resistance in Helicobacter pylori (H. pylori) is the major factor for eradication failure. Molecular tests including fluorescence in situ hybridization, PCR-restriction fragment length polymorphism, and dual priming oligonucleotide-PCR (DPO-PCR) play critical roles in the detection of antibiotic susceptibility; however, limited knowledge is known about application of multiple genetic analysis system (MGAS) in the area of H. pylori identification and antibiotics resistance detection.The aim of this study is to determine the antibiotics resistance using different molecular tests and evaluate the treatment outcomes of E-test-based genotypic resistance.A total of 297 patients with dyspepsia complaint were recruited for gastroscopies. Ninety patients with H. pylori culture positive were randomly divided into 2 groups (test group and control group). E-test, general PCR, and MGAS assay were performed in test group. Patients in control group were treated with empirical therapy (rabeprazole + bismuth potassium citrate + amoxicillin [AMX] + clarithromycin [CLR]), whereas patients in test group received quadruple therapy based on E-test results twice daily for 14 consecutive days. The eradication effect of H. pylori was confirmed by 13C-urea breath test after at least 4 weeks when treatment was finished.Rapid urease test showed 46.5% (128/297) patients with H. pylori infection, whereas 30.3% (90/297) patients were H. pylori culture positive. E-test showed that H. pylori primary resistance rate to CLR, AMX, metronidazole, tetracycline, and levofloxacin (LVX) was 40.0% (18/45), 4.4% (2/45), 53.3% (24/45), 0% (0/45), and 55.6% (25/45), respectively. In addition, there are many multidrug resistant (MDR) phenotypes, and the MDR strains have higher minimum inhibitory concentration than their single-drug resistant counterparts. Considering E-test as the reference test, the sensitivities of general PCR and MGAS in detecting CLR resistance were 83.3% (15/18) and 94.4% (17/18), whereas in detecting LVX resistance were 100% (25/25) and 83.3% (15/18), respectively. Finally, the eradication rate in test group was significantly higher than that in control group as demonstrated by intention-to-treat analysis and per-protocol analysis.MGAS is a promising assay for H. pylori identification and antibiotic susceptibility testing. Phenotypic resistance-guided quadruple therapy showed a high efficacy in treating patients with H. pylori infection.

Aims/IntroductionIn previous studies, hydrogen gas (H2) administration has clearly shown effectiveness in inhibiting diabetes. Here, we evaluated whether subcutaneous injection of H2 shows enhanced efficacy against type 2 diabetes mellitus induced in mice by a high‐fat diet and low‐dose streptozotocin treatment.Material and MethodsH2 was injected subcutaneously at a dose of 1 mL/mouse/week for 4 weeks. Type 2 diabetes mellitus‐associated parameters were then evaluated to determine the effectiveness of subcutaneous H2 administration.ResultsThe bodyweight of H2‐treated mice did not change over the course of the experiment. Compared with the untreated control animals, glucose, insulin, low‐density lipoprotein and triglyceride levels in the serum were significantly lower in treated mice, whereas high‐density lipoprotein cholesterol in the serum was significantly higher. Glucose tolerance and insulin sensitivity were both improved in H2‐treated mice. Diabetic nephropathy analysis showed significant reductions in urine volume, urinary total protein and β2‐microglobulin, kidney/bodyweight ratio, and kidney fibrosis associated with subcutaneous injection of H2.ConclusionsSubcutaneous injection of H2 significantly improves type 2 diabetes mellitus and diabetic nephropathy‐related outcomes in a mouse model, supporting further consideration of subcutaneous injection as a novel and effective route of clinical H2 administration.

HMGS is an effective method for H. pylori identification and analysis of virulence and drug resistance.

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.

BackgroundMeningitis and encephalitis (ME) are central nervous system (CNS) infections mainly caused by bacteria, mycobacteria, fungi, viruses, and parasites that result in high morbidity and mortality. The early, accurate diagnosis of pathogens in the cerebrospinal fluid (CSF) and timely medication are associated with better prognosis. Conventional methods, such as culture, microscopic examination, serological detection, CSF routine analysis, and radiological findings, either are time‐consuming or lack sensitivity and specificity.MethodsTo address these clinical needs, we developed an advanced fragment analysis (AFA)‐based assay for the multiplex detection of 22 common ME pathogens, including eight viruses, 11 bacteria, and three fungi. The detection sensitivity of each target was evaluated with a recombinant plasmid. The limits of detection of the 22 pathogens ranged from 15 to 120 copies/reaction. We performed a retrospective study to analyze the pathogens from the CSF specimens of 170 clinically diagnosed ME patients using an AFA‐based assay and compared the results with culture (bacteria and fungi), microscopic examination (fungi), polymerase chain reaction (PCR) (Mycobacterium tuberculosis), and Sanger sequencing (virus) results.ResultsThe sensitivity of the AFA assay was 100% for 10 analytes. For Cryptococcus neoformans, the sensitivity was 63.6%. The overall specificity was 98.2%. The turnaround time was reduced to 4‐6 hours from the 3‐7 days required using conventional methods.ConclusionsIn conclusion, the AFA‐based assay provides a rapid, sensitive, and accurate method for pathogen detection from CSF samples.

BackgroundCulture-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.ResultsDP-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.ConclusionsDP-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.Electronic supplementary materialThe online version of this article (10.1186/s13099-018-0264-7) contains supplementary material, which is available to authorized users.

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.

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