Establishment of a Multiplex Detection Method for Common Bacteria in Blood Based on Human Mannan-Binding Lectin Protein-Conjugated Magnetic Bead Enrichment Combined with Recombinase-Aided PCR Technology.

Objective: Recombinase-aided polymerase chain reaction (RAP) is a sensitive, single-tube, two-stage nucleic acid amplification method. This study aimed to develop an assay that can be used for the early diagnosis of three types of bacteremia caused by Staphylococcus aureus (SA), Pseudomonas aeruginosa (PA), and Acinetobacter baumannii (AB) in the bloodstream based on recombinant human mannan-binding lectin protein (M1 protein)-conjugated magnetic bead (M1 bead) enrichment of pathogens combined with RAP. Methods: Recombinant plasmids were used to evaluate the assay sensitivity. Common blood influenza bacteria were used for the specific detection. Simulated and clinical plasma samples were enriched with M1 beads and then subjected to multiple recombinase-aided PCR (M-RAP) and quantitative PCR (qPCR) assays. Kappa analysis was used to evaluate the consistency between the two assays. Results: The M-RAP method had sensitivity rates of 1, 10, and 1 copies/µL for the detection of SA, PA, and AB plasmids, respectively, without cross-reaction to other bacterial species. The M-RAP assay obtained results for < 10 CFU/mL pathogens in the blood within 4 h, with higher sensitivity than qPCR. M-RAP and qPCR for SA, PA, and AB yielded Kappa values of 0.839, 0.815, and 0.856, respectively ( P < 0.05). Conclusion: An M-RAP assay for SA, PA, and AB in blood samples utilizing M1 bead enrichment has been developed and can be potentially used for the early detection of bacteremia.

A Multiplex Recombinase-Aided qPCR Assay for Highly Sensitive and Rapid Detection of khe, blaKPC -2, and blaNDM -1 Genes in Klebsiella pneumoniae.

OBJECTIVE: This study aimed to establish a highly sensitive and rapid single-tube, two-stage, multiplex recombinase-aided qPCR (mRAP) assay to specifically detect the khe, blaKPC-2, and blaNDM-1 genes in Klebsiella pneumoniae. METHODS: mRAP was carried out in a qPCR instrument within 1 h. The analytical sensitivities of mRAP for khe, blaKPC-2, and blaNDM-1 genes were tested using recombinant plasmids and dilutions of reference strains. A total of 137 clinical isolates and 86 sputum samples were used to validate the clinical performance of mRAP. RESULTS: mRAP achieved the sensitivities of 10, 8, and 14 copies/reaction for khe, blaKPC-2, and blaNDM-1 genes, respectively, superior to qPCR. The Kappa value of qPCR and mRAP for detecting khe, blaKPC-2, and blaNDM-1 genes was 1, 0.855, and 1, respectively (p < 0.05). CONCLUSION: mRAP is a rapid and highly sensitive assay for potential clinical identification of khe, blaKPC-2, and blaNDM-1 genes in K. pneumoniae.

Clinical and endoscopic features of primary small bowel lymphoma: a single-center experience from mainland China.

Objective: The worldwide incidence of primary small intestinal lymphoma (PSIL) is increasing. However, little is known about the clinical and endoscopic characteristics of this disease. The aim of this study was to investigate the clinical and endoscopic data of patients with PSIL, with the goal of enhancing our understanding of the disease, improving diagnostic accuracy, and facilitating more accurate prognosis estimation. Methods: Ninety-four patients diagnosed with PSIL were retrospectively studied at Qilu Hospital of Shandong University between 2012 and 2021. The clinical data, enteroscopy findings, treatment modalities, and survival times were collected and analyzed. Results: Ninety-four patients (52 males) with PSIL were included in this study. The median age of onset was 58.5 years (range: 19-80 years). Diffuse large B-cell lymphoma (n=37) was the most common pathological type. Abdominal pain (n=59) was the most frequent clinical presentation. The ileocecal region (n=32) was the most commonly affected site, and 11.7% of patients had multiple lesions. At the time of diagnosis, the majority of patients (n=68) were in stages I-II. A new endoscopic classification of PSIL was developed, including hypertrophic type, exophytic type, follicular/polypoid type, ulcerative type, and diffusion type. Surgery did not show a significant increase in overall survival; chemotherapy was the most commonly administered treatment. T-cell lymphoma, stages III-IV, "B" symptoms, and ulcerative type were associated with poor prognosis. Conclusion: This study provides a comprehensive analysis of the clinical and endoscopic features of PSIL in 94 patients. This highlights the importance of considering clinical and endoscopic characteristics for accurate diagnosis and prognosis estimation during small bowel enteroscopy. Early detection and treatment of PSIL is associated with a favorable prognosis. Our findings also suggest that certain risk factors, such as pathological type, "B" symptoms, and endoscopic type, may affect the survival of PSIL patients. These results underscore the need for careful consideration of these factors in the diagnosis and treatment of PSIL.

Effects of antithrombotic therapy on bleeding after endoscopic sphincterotomy: A systematic review and meta-analysis.

Background and study aims Bleeding is a common complication of following endoscopy sphincterotomy (EST), and antithrombotic therapy use during the procedure often increases risk of it. Although several guidelines have been released regarding the use of antithrombotic agents during EST, many issues about it remain controversial. We carried out a systematic review and meta-analysis to evaluate the effect of antithrombotic medication on the risk of EST bleeding. Methods A structured literature search was carried out in Web of Science, EMBASE, PubMed, and Cochrane Library databases. RevMan 5.2 was used for meta-analysis to investigate the rate of post-EST bleeding. Results Seven retrospective articles were included. Compared with patients who had never taken antithrombotic drugs, patients who discontinued antithrombotic drugs 1 day before the procedure had a significantly increased risk of post-EST bleeding (OR, 1.95; 95 %CI, 1.57-2.43), particularly for severe bleeding (OR, 1.83; 95 %CI, 1.44-2.34). In addition, compared with patients who discontinued antithrombotic therapy for at least 1 day, patients who continued taking antithrombotic drugs did have an increased risk of post-EST bleeding (OR, 0.70; 95 %CI, 0.40-1.23). Conclusions The use of antithrombotic drugs may increase the bleeding rate of EST, but discontinuing therapy 1 day before endoscopy does not significantly reduce the bleeding rate.

Development of a duplex recombinase-aided amplification assay for direct detection of Mycoplasma pneumoniae and Chlamydia trachomatis in clinical samples.

BACKGROUND: Pneumonia caused by Mycoplasma pneumoniae is common in the elderly and children, and pneumonia caused by Chlamydia trachomatis is prevalent in newborns. This study aimed to establish a rapid, sensitive, and simple method for the direct detection of M. pneumoniae and C. trachomatis in clinical samples without DNA extraction. METHODS: We established a duplex recombinase-aided amplification (RAA) assay with the RNAseP gene as an internal control for detecting the P1 gene of M. pneumoniae and the ORF8 gene of C. trachomatis, respectively. The results were obtained at 39 °C within 15-20 min. A total of 130 clinical samples suspected of M. pneumoniae or C. trachomatis infection were collected and tested by duplex RAA and PCR. DNA extracted via a commercial kit or treated with a nucleic acid-releasing agent was used and compared, respectively. Standard recombinant plasmids were used to test the sensitivity of the duplex RAA assay. In addition, other similar common pathogens were used to verify the specificity of the duplex RAA assay. RESULTS: The sensitivity of the duplex RAA assay for detecting M. pneumoniae and C. trachomatis was 10 copies/µL using recombinant plasmids. Compared with PCR, the sensitivity and specificity of duplex RAA assays for M. pneumoniae and C. trachomatis was 100% using clinical DNA samples extracted using a commercial kit and a nucleic acid-releasing agent, and the Kappa value was 1. CONCLUSION: The advantages of this duplex RAA assay include high sensitivity and specificity, short duration, and simple extraction steps, with potential for use in the on-site detection of M. pneumoniae and C. trachomatis in resource-limited settings.

Detection of low-load Epstein-Barr virus in blood samples by enriched recombinase aided amplification assay.

Epstein-Barr virus (EBV), a common human γ-herpesvirus, infects more than 90% of adults worldwide. The purpose of this study was to establish a novel EBV detection method by combining the recombinase aided amplification (RAA) assay with an initial enrichment step that utilizes magnetic beads coated with a recombinant human mannan-binding lectin (rhMBL, M1 protein). An M1 protein-protein A magnetic bead complex (M1 beads) was prepared and used to achieve separation and enrichment of EBV from blood. After nucleic acid extraction, DNA was amplified by RAA. Using 388 whole blood samples and 1 serum sample, we explored the specificity, sensitivity and applicability of the newly developed detection method and compared it with commercial quantitative real-time polymerase chain reaction (qPCR) following M1 bead enrichment, traditional qPCR and traditional RAA. After enrichment, the positivity rate of EBV was increased from 15.94% to 17.74% by RAA (P < 0.05) and from 7.20% to 15.17% by qPCR (P < 0.05). The viral loads after enrichment were increased by 1.13 to 23.19-fold (P < 0.05). Our data demonstrates that an RAA assay incorporating M1 bead enrichment is a promising tool for detecting low EBV viral loads in blood samples that will facilitate an early response to EBV infection.

HYAL-1-induced autophagy facilitates pancreatic fistula for patients who underwent pancreaticoduodenectomy.

The main mechanism of hyaluronidase 1(HYAL-1) in the development of postoperative pancreatic fistula (POPF) after pancreatoduodenectomy (PD) was unknown. In this study, a comprehensive inventory of pre-, intra-, and postoperative clinical and biological data of two cohorts (62 pancreatic cancer [PCa] and 111 pancreatic ductal adenocarcinoma [PDAC]) which could induce POPF were retrospectively analyzed. Then, a total of 7644 genes correlated with HYAL-1 was predicted in PDAC tissues and the enriched pathway, kinase targets and biological process of those correlated genes were evaluated. Finally, a mouse pancreatic fistula (PF) model was first built and in vitro studies were performed to investigate the effects of HYAL-1 on PF progression. Our data indicated that preoperative serum HYAL-1 level, pancreatic fibrosis score, and pancreatic duct size were valuable factors for detecting POPF of Grade B and C. The serum HYAL-1 level of 2.07 mg/ml and pancreatic fibrosis score of 2.5 were proposed as the cutoff values for indicating POPF. The bioinformatic analysis and in vitro and in vivo studies demonstrated that HYAL-1 facilitates pancreatic acinar cell autophagy via the dephosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK) and signal transducers and activators of transcription 3 (STAT3) signaling pathways, which exacerbate pancreatic secretion and inflammation. In summary, the preoperative serum HYAL-1 was a significant predictor for POPF in patients who underwent PD. Tumor-induced HYAL-1 is one of core risk in accelerating PF and then promoting pancreatic secretion and acute inflammation response through the AMPK and STAT3-induced autophagy.

Plasma D-Dimer Level Is an Early Predictor of Severity of Acute Pancreatitis Based on 2012 Atlanta Classification.

BACKGROUND Acute pancreatitis (AP) is a common digestive disorder. Its management depends on the severity; therefore, it is essential to stratify AP patients early. D-dimer, a coagulation indicator, appears to be associated with the pathogenesis of AP. The aim of this study was to evaluate D-dimer as an early predictor of the severity of AP. MATERIAL AND METHODS This was a single-center retrospective study of 1260 patients diagnosed based on the revised Atlanta classification. Only patients hospitalized within 24 h of onset were included, and 334 patients were enrolled. Blood was collected at admission and 3 times within 48 h of admission. Values at admission and average of the 3 blood samples were evaluated by univariate and multivariate analyses. Furthermore, the area under the receiver-operating characteristic curve (AUC) was used to estimate the validity of the predictor and to define optimal cut-off points for prediction. RESULTS We found that 53.3% of the patients had mild AP (MAP), 24.3% had moderately severe AP (MSAP), and 22.4% had severe AP (SAP). D-dimer at admission and the average D-dimer could distinguish MAP patients from MSAP and SAP patients, with cut-off values of 3.355 mg/L and 4.868 mg/L, respectively. No difference in the parameters at admission was observed in multivariate analysis in distinguishing SAP from MSAP, but the average D-dimer level was significantly different with a cut-off value of 7.268 mg/L by comparing Ranson score, APACHE II score, and D-dimer level. CONCLUSIONS The average value of D-dimer levels could be used as a predictor of severity of AP. In general, patients with an average D-dimer level <4.868 could be diagnosed with MAP, >7.268 would develop into SAP, and between 4.868 and 7.268 would be MSAP.

RB1CC1-enhanced autophagy facilitates PSCs activation and pancreatic fibrogenesis in chronic pancreatitis.

Chronic pancreatitis (CP) is described as a progressive fibro-inflammatory disorder of the exocrine disease, which eventually leads to damage of the gland. Excessive activation of pancreatic stellate cells (PSCs) is a critical participant in the initiation of CP. Autophagy is involved in multiple degeneration and inflammation in acute pancreatitis and CP. In our study, we report that retinoblastoma coiled coil protein 1 (RB1CC1) expression and the autophagic level are elevated in activated PSCs. RB1CC1 is positively correlated with pancreatic fibrogenesis in tissues and plasma of CP patients. Knockdown of RB1CC1 restrains alpha smooth muscle actin (α-SMA) and collagen expressions, and autophagy in activated PSCs in vitro. Furthermore, we show that RB1CC1 induces PSC activation via binding to ULK1 promoter and the direct interaction with ULK1 protein. These suppress ULK1 expression and its kinase activity. In mice, knockdown of RB1CC1 blocks autophagy and then inhibits the pancreatic duct ligation-induced pancreatic fibrosis. Consequently, our study highlights that RB1CC1-mediated autophagy is a key event for the activation of PSCs. Inhibition of RB1CC1 alleviates autophagy, which plays a critical role in anti-fibrotic activation in PSCs and CP progression. RB1CC1 could be a novel strategy for the treatment of pancreatic fibrosis.

Feasibility of balanced steady-state free precession sequence at 1.5T for the evaluation of hepatic steatosis in obese children and adolescents.

OBJECTIVES: To determine the feasibility of balanced steady-state free precession (b-SSFP) imaging for measuring hepatic steatosis in obese children and adolescents, using proton magnetic resonance spectroscopy (1H MRS) as reference standard. METHODS: 182 obese Chinese paediatric patients underwent conventional T1-weighted dual echo MRI, 1H MRS and b-SSFP imaging for non-invasive assessment of hepatic steatosis. RESULTS: There was a strong positive correlation between liver fat fraction (FF) on T1-weighted dual echo MRI and 1H MRS-determined liver fat content (LFC) (r = 0.964, p < .001), and a strong negative correlation between the ratio of liver signal intensity (SI) to spleen SI (L/S) on b-SSFP and LFC (r = -0.896, p < .001). ROC curve analysis based on a diagnostic threshold of 1H MRS-determined LFC >50 mg/g (>5 % by wet weight) showed areas under the curves for FF and L/S at 0.989 (0.976-1.000) and 0.926 (0.888-0.964), respectively. Optimal FF and L/S cut-off values identified patients with hepatic steatosis with 97.9 % and 86.5 % sensitivity and 93.4 % and 93.4 % specificity, respectively. CONCLUSIONS: Following further validation, b-SSFP at 1.5T has potential as a feasible technique for evaluation of hepatic steatosis in obese paediatric patients with limited breath-holding capacity. KEY POINTS: • L/S on b-SSFP images closely correlated with 1 H MRS-determined LFC. • b-SSFP has high diagnostic accuracy for hepatic steatosis in obese children. • 100% of obese paediatric subjects are imaged successfully using b-SSFP sequence. • b-SSFP has potential to evaluate hepatic steatosis in children with poor breath-hold.

Synthesis of a Ni2P/Ni12P5 bi-phase nanocomposite for the efficient catalytic reduction of 4-nitrophenol based on the unique n-n heterojunction effects.

A novel heterostructure catalyst of Ni2P/Ni12P5 has been fabricated through a simple solvothermal method by modifying the molar ratio of the initial raw materials. The products are characterized by X-ray powder diffraction (XRD), field emission scanning electron microscopy (FE-SEM), high-resolution transmission electron microscopy (HRTEM), nitrogen adsorption and X-ray photoelectron spectroscopy (XPS). It is found that the two phases, Ni2P and Ni12P5, are interlaced with one another in the as-formed nanocomposite, resulting in more interfaces. The bi-phase catalyst exhibits a markedly enhanced catalytic activity in the reduction of 4-nitrophenol, as compared to that of single Ni2P or Ni12P5. The enhanced catalytic activity can be attributed to the unique n-n series effects, which result in the increased ease of electron transfer over the Ni2P/Ni12P5 bi-phase catalyst.