Development and evaluation of a rapid visual loop-mediated isothermal amplification assay for the tcdA gene in Clostridioides difficile detection.

Background: The tcdA gene codes for an important toxin produced by Clostridioides difficile (C. difficile), but there is currently no simple and cost-effective method of detecting it. This article establishes and validates a rapid and visual loop-mediated isothermal amplification (LAMP) assay for the detection of the tcdA gene. Methods: Three sets of primers were designed and optimized to amplify the tcdA gene in C. difficile using a LAMP assay. To evaluate the specificity of the LAMP assay, C. difficile VPI10463 was used as a positive control, while 26 pathogenic bacterial strains lacking the tcdA gene and distilled water were utilized as negative controls. For sensitivity analysis, the LAMP assay was compared to PCR using ten-fold serial dilutions of DNA from C. difficile VPI10463, ranging from 207 ng/µl to 0.000207 pg/µl. The tcdA gene of C.difficile was detected in 164 stool specimens using both LAMP and polymerase chain reaction (PCR). Positive and negative results were distinguished using real-time monitoring of turbidity and chromogenic reaction. Results: At a temperature of 66 °C, the target DNA was successfully amplified with a set of primers designated, and visualized within 60 min. Under the same conditions, the target DNA was not amplified with the tcdA12 primers for 26 pathogenic bacterial strains that do not carry the tcdA gene. The detection limit of LAMP was 20.700 pg/µl, which was 10 times more sensitive than that of conventional PCR. The detection rate of tcdA in 164 stool specimens using the LAMP method was 17% (28/164), significantly higher than the 10% (16/164) detection rate of the PCR method (X2 = 47, p < 0.01). Conclusion: LAMP method is an effective technique for the rapid and visual detection of the tcdA gene of C. difficile, and shows potential advantages over PCR in terms of speed, simplicity, and sensitivity. The tcdA-LAMP assay is particularly suitable for medical diagnostic environments with limited resources and is a promising diagnostic strategy for the screening and detection of C. difficile infection in populations at high risk.

Saccharomyces boulardii combined with triple therapy alter the microbiota in the eradication of Helicobacter pylori infection.

To assess the effectiveness and safety of combining Saccharomyces boulardii powder with triple therapy as a primary approach for eradicating H. pylori infection, a total of 144 patients who tested positive for H. pylori and diagnosed with non-ulcer dyspepsia underwent endoscopy at two national centers between June 2017 and March 2019 were included. The patients were categorized into three groups using a subsection randomization method and received initial H. pylori eradication treatments. Microbial composition, eradication rates, symptom alleviation, and adverse reactions were monitored on the 14th and 44th days post-treatment. According to PP analysis showed the eradication rates for the SRAC group was 75%, BRAC was 93.18% and RAC was 65.2%. Group BRAC exhibited a marginally higher eradication rate compared to other groups. However, patients receiving Saccharomyces boulardii treatment exhibited an overall reduction in initial dyspepsia symptoms by the end of the treatment period. When employed as a primary strategy, the combination of Saccharomyces boulardii powder with triple therapy displayed notable efficacy and smaller gastrointestinal side effects in eradicating initial H. pylori infections among non-ulcer dyspepsia patients. Moreover, this approach demonstrated advantages in alleviating symptoms, exhibited favorable tolerance, and maintained a high level of clinical safety.

Upadacitinib for refractory ulcerative colitis with primary nonresponse to infliximab and vedolizumab: A case report.

BACKGROUND: Many patients with ulcerative colitis (UC) do not respond well to, or tolerate conventional and biological therapies. There is currently no consensus on the treatment of refractory UC. Studies have demonstrated that the selective Janus kinase 1 inhibitor upadacitinib, a small-molecule drug, is effective and safe for treating UC. However, no studies have revealed that upadacitinib is effective in treating refractory UC with primary nonresponse to infliximab and vedolizumab. CASE SUMMARY: We report the case of a 44-year-old male patient with a chief complaint of bloody diarrhoea with mucus and pus, in addition to dizziness. The patient had recurrent disease after receiving mesalazine, prednisone, azathioprine, infliximab and vedolizumab over four years. Based on the endoscopic findings and pathological biopsy, the patient was diagnosed with refractory UC. In particular, the patient showed primary nonresponse to infliximab and vedolizumab. Based on the patient's history and recurrent disease, we decided to administer upadacitinib. During hospitalisation, the patient was received upadacitinib under our guidance. Eight weeks after the initiation of upadacitinib treatment, the patient's symptoms and endoscopic findings improved significantly. No notable adverse reactions have been reported to date. CONCLUSION: Our case report suggests that upadacitinib may represent a valuable strategy for treating refractory UC with primary nonresponse.

A probiotic bi-functional peptidoglycan hydrolase sheds NOD2 ligands to regulate gut homeostasis in female mice.

Secreted proteins are one of the direct molecular mechanisms by which microbiota influence the host, thus constituting a promising field for drug discovery. Here, through bioinformatics-guided screening of the secretome of clinically established probiotics from Lactobacillus, we identify an uncharacterized secreted protein (named LPH here) that is shared by most of these probiotic strains (8/10) and demonstrate that it protects female mice from colitis in multiple models. Functional studies show that LPH is a bi-functional peptidoglycan hydrolase with both N-Acetyl-ß-D-muramidase and DL-endopeptidase activities that can generate muramyl dipeptide (MDP), a NOD2 ligand. Different active site mutants of LPH in combination with Nod2 knockout female mice confirm that LPH exerts anti-colitis effects through MDP-NOD2 signaling. Furthermore, we validate that LPH can also exert protective effects on inflammation-associated colorectal cancer in female mice. Our study reports a probiotic enzyme that enhances NOD2 signaling in vivo in female mice and describes a molecular mechanism that may contribute to the effects of traditional Lactobacillus probiotics.

Gut microbial DL-endopeptidase alleviates Crohn's disease via the NOD2 pathway.

The pattern-recognition receptor NOD2 senses bacterial muropeptides to regulate host immunity and maintain homeostasis. Loss-of-function mutations in NOD2 are associated with Crohn's disease (CD), but how the variations in microbial factors influence NOD2 signaling and host pathology is elusive. We demonstrate that the Firmicutes peptidoglycan remodeling enzyme, DL-endopeptidase, increased the NOD2 ligand level in the gut and impacted colitis outcomes. Metagenomic analyses of global cohorts (n = 857) revealed that DL-endopeptidase gene abundance decreased globally in CD patients and negatively correlated with colitis. Fecal microbiota from CD patients with low DL-endopeptidase activity predisposed mice to colitis. Administering DL-endopeptidase, but not an active site mutant, alleviated colitis via the NOD2 pathway. Therapeutically restoring NOD2 ligands with a DL-endopeptidase-producing Lactobacillus salivarius strain or mifamurtide, a clinical analog of muramyl dipeptide, exerted potent anti-colitis effects. Our study suggests that the depletion of DL-endopeptidase contributes to CD pathogenesis through NOD2 signaling, providing a therapeutically modifiable target.

The appropriate cutoff gastric pH value for Helicobacter pylori eradication with bismuth-based quadruple therapy.

BACKGROUND: This study aimed to investigate whether an increased proton pump inhibitor (PPI) dose enhanced the efficacy of Helicobacter pylori (H. pylori) eradication and determine the appropriate cutoff intragastric pH value that could predict H. pylori eradication with bismuth-based quadruple therapy. MATERIALS AND METHODS: A total of 207 H. pylori infected, treatment naive patients were enrolled in this prospective, open-label, randomized controlled trial. Patients were randomly allocated into Eso40-group (esomeprazole 40 mg bid) and Eso20-group (esomeprazole 20 mg bid), and their CYP2C19 genotyping status was assessed. The 24-h intragastric pH monitoring on day 7 was performed, and percentage of time gastric pH ≥ 3, ≥4, ≥5, and ≥6 (pH holding time ratios; HTRs) were measured. H. pylori eradication was evaluated using 13 C-urea breath test. RESULTS: No significant difference in the eradication rates was observed between two groups. The median 24-h intragastric pH value was not significant different between two groups but the Eso40 Group had a significant higher pH4 HTRs (91.11% [95%CI: 87.50%-95.83%] vs. 95.83% [95.83%-100%]; p = .002). Additionally, the median 24-h intragastric pH value showed significantly difference between two groups in EM genotype (Eso20 Group 6.00 [95%CI; 5.75-6.15] vs. Eso40 Group 6.30 [6.05-6.30]; p = .019). Similar results were observed in pH4 HTRs. There were significant differences in intragastric pH value (6.10 [95%CI: 4.40-7.00] vs. 5.65 [4.85-5.95], p = .038) and in pH4 HTRs (96% [95%CI: 92.00%-96.00%] vs. 87.5% [67.00%-100.0%], p = .019) between eradication-successful and eradication-failed patients. Statistical analysis suggested that the median intragastric pH = 5.7 could identify the success of H. pylori eradication. CONCLUSIONS: Bismuth-based quadruple therapy resulted in high H. pylori eradication rates either in PPI standard or double doses. Double dose of esomeprazole is associated with better intragastric acid suppression. A median 24-h intragastric pH of 5.7 could be appropriate cutoff value for predicting the successful H. pylori eradication.

Identification of quality control markers in Suhuang antitussive capsule based on HPLC-PDA fingerprint and anti-inflammatory screening.

Suhuang antitussive capsule (SH), one of traditional Chinese patent medicines, has been widely used for treating cough variant asthma and postinfectious cough in clinic. The objective of this work is to identify the characteristic and active ingredients as the quality control markers for SH based on high performance liquid chromatography with photodiode array detector (HPLC-PDA) fingerprint and screening of anti-inflammatory components. Similarity analysis (SA), hierarchical clustering analysis (HCA) and principal component analysis (PCA) were used to evaluate 16 different batches of SH. 13 compounds accounting for 36% of the total components in the fingerprint were identified and semi-quantitatively analyzed, which anti-inflammatory activity was tested with the in vitro assay. The results showed that the established chemical fingerprint could clearly distinguish different batches of SH by SA, HCA, and PCA analysis. Furthermore, four known compounds (chlorogenic acid, schisandrin, angeloylgomisin H and praeruptorin A) were screened out to be the most discriminant variables, which could be applied to quality control of SH by quantitative analysis. The semi-quantitative results showed that six compounds were major components, i.e. arctiin (10.28 ±â€¯3.18 mg/g), ephedrine (9.26 ±â€¯1.58 mg/g), schisandrin (3.09 ±â€¯0.83 mg/g), pseudoephedrine (2.34 ±â€¯1.04 mg/g), schisandrin B (1.48 ±â€¯0.16 mg/g), and 1-caffeoylquinic acid (1.36 ±â€¯0.42 mg/g). The anti-inflammatory results showed that SH extract, praeruptorin A, schisandrin, arctigenin and pseudoephedrine could significantly inhibit inflammatory mediator NO production in LPS-stimulated RAW264.7 macrophages. These findings indicated that praeruptorin A, schisandrin, arctiin and pseudoephedrine could be proposed as the quality control markers for SH.

[A new phenolic glycoside from honey-fried Eriobotrya japonica].

A total of twelve compounds were isolated from the ethyl acetate of the water extract of honey-fried Eriobotrya japonica through column chromatography over silica gel,Sephadex LH-20,RP-18,and preparative HPLC. Their structures were established by MS,1 D NMR and 2 D NMR data as japonicanoside A( 1),nerolidol-3-O-α-L-rhamnopyranosyl-( 1→2)-ß-D-glucopyranoside( 2),nerolidol-3-O-α-L-rhamnopyranosyl-( l→4)-α-L-rhamnopyranosyl-( 1 → 2)-[α-L-( 4-trans-feruloyl)-rhamnopyranosyl-( 1 → 6) ]-ß-D-glucopyranoside( 3),( +)-catechin( 4),(-)-epicatechin( 5),kaempferol 3-O-α-L-rhamnopyranoside( 6),quercitrin( 7),quercetin-3-O-ß-D-galactopyranoside( 8),quercetin-3-O-ß-glucopyranoside( 9),vanillin( 10),protocatechuic aldehyde( 11),and maltol( 12). Among them,1 is a new phenolic glycoside.

Fusobacterium nucleatum Aggravates the Progression of Colitis by Regulating M1 Macrophage Polarization via AKT2 Pathway.

Disordered intestinal flora and discordant immune response are associated with the development of ulcerative colitis (UC). Recent work has described the ability of macrophages to undergo repolarization toward a proinflammatory M1 or anti-inflammatory M2 phenotype in response to particular bacterium-derived signals. Fusobacterium nucleatum (F. nucleatum, Fn) is a species of intestinal commensal bacteria with potential pathogenicity, but its association with UC and how it may contribute to progression of UC is largely unknown. In this study, we provide new evidence that F. nucleatum accumulated heavily in the intestine of UC patients and was accompanied by the secretion of IFN-γ and the skewing of M1 macrophages. Mechanistically, our data showed that F. nucleatum aggravated dextran sodium sulfate (DSS)-induced colitis in the production of Th1-related cytokines IFN-γ through the AKT2 signaling pathway in vitro and in vivo. To further confirm the disease-relevance of these shifts in macrophage repolarization in response to F. nucleatum, stimulated bone marrow-derived macrophages (BMDMs) were transferred into recipient mice with DSS colitis. This transfer resulted in increased disease activity and inflammatory cytokine production. Taken together, we show clearly that F. nucleatum can promote the progression of UC via proinflammatory M1 macrophage skewing, and targeting F. nucleatum or AKT2 signaling may be a viable means of blocking development of UC.

Half-dose clarithromycin-containing bismuth quadruple therapy is effective and economical in treating Helicobacter pylori infection: A single-center, open-label, randomized trial.

BACKGROUND: Clarithromycin-containing bismuth quadruple therapy has been recommended as the first-line therapy for H pylori infection in China. However, its expensive cost and high antibiotic-related adverse reactions are always haunting us. To find a safer, more cost-effective, and high eradicative strategy for Helicobacter treatment, we investigated the efficacy of 14-day bismuth quadruple therapy and different doses of clarithromycin in the first-line treatment. METHOD: A total of 210 patients with H pylori infection were recruited and randomly assigned to half-dose clarithromycin group (esomeprazole 20 mg bid, amoxicillin 1 g bid, clarithromycin 250 mg bid, and bismuth potassium citrate 0.6 g bid) for 14 days or standard-dose clarithromycin group (esomeprazole 20 mg bid, amoxicillin 1 g bid, clarithromycin 500 mg bid, and bismuth potassium citrate 0.6 g bid) for 14 days. A 13 C-urea breath test (13 C-UBT) was performed at least 4 weeks after treatment. The eradication rate of H pylori, the incidence of side effects, and the cost-effectiveness of regimens were evaluated in this study. RESULTS: The eradication frequencies were 86.67% for both groups in the intention-to-treat analysis, while the per-protocol eradication rates were 91% vs. 91.92% (p=0.817). The incidence of adverse events was higher in standard dose group (54.21% vs. 34.29%; p=0.004), especially bitter taste symptom. There was a higher level of costs per person associated with the standard-dose group as compared with half-dose group (ï¿¥804.3 vs ï¿¥654.36). The cost-effectiveness ratio of the half dose was less than that of the standard dose (7.55 vs 9.16 CNY per percent). CONCLUSIONS: A 14-day half-dose clarithromycin-containing bismuth quadruple regimen is as effective as the standard bismuth quadruple therapy at eradicating H pylori, which is better tolerated and more economical. (ChiCTR-ROC-15007406).

Prognostic value of PIVKA-II in hepatocellular carcinoma patients receiving curative ablation: A systematic review and meta-analysis.

BACKGROUND: Several studies have been conducted to evaluate the prognostic value of prothrombin induced by vitamin K absence-II (PIVKA-II) overexpression in hepatocellular carcinoma patients treated with curative ablation. However, the results remain controversial. The purpose of this meta-analysis was to explore the correlation between PIVKA-II expression and survival outcomes in these patients. METHODS: We performed a systematic literature search in PubMed, EMBASE, Medline, Cochrane Library, and Web of Science to identify the relevant articles investigating the prognostic value of PIVKA-II in patients with hepatocellular carcinoma. Combined hazard ratios (HR) and their 95% confidence intervals (CI) for overall survival and recurrence-free survival were calculated as the analysis endpoints. RESULTS: A total of 15 cohorts encompassing 5647 patients were included. The results indicated that elevated PIVKA-II was significantly associated with poorer overall survival (HR 1.59; 95% CI 1.40, 1.82; P < 0.001) and recurrence-free survival (HR 1.76; 95% CI 1.42, 2.17; P < 0.001). Similar results were observed in the subgroup analysis based on sample size, analytical method, treatment modality, and cut-off value. CONCLUSIONS: This meta-analysis suggests that elevated PIVKA-II is a predictor of unfavorable overall survival and recurrence-free survival in hepatocellular carcinoma patients receiving curative ablation. More rigorous studies are warranted to confirm the clinical utility of PIVKA-II in determining hepatocellular carcinoma prognosis.