Impact of mixed-infection rate of clarithromycin-susceptible and clarithromycin-resistant Helicobacter pylori strains on the success rate of clarithromycin-based eradication treatment.

BACKGROUND: Clarithromycin (CAM) resistance is a major contributor to the failure to eradicate Helicobacter pylori (H. pylori). The mixed-infection ratio of CAM-susceptible and CAM-resistant H. pylori strains differs among individuals. Pyrosequencing analysis can be used to quantify gene mutations at position each 2142 and 2143 of the H. pylori 23S rRNA gene in intragastric fluid samples. Herein, we aimed to clarify the impact of the rate of mixed infection with CAM-susceptible and CAM-resistant H. pylori strains on the success rate of CAM-containing eradication therapy. MATERIALS AND METHODS: Sixty-four H. pylori-positive participants who received CAM-based eradication therapy, also comprising vonoprazan and amoxicillin, were enrolled in this prospective cohort study. Biopsy and intragastric fluid samples were collected during esophagogastroduodenoscopy. H. pylori culture and CAM-susceptibility tests were performed on the biopsy samples, and real-time PCR and pyrosequencing analyses were performed on the intragastric fluid samples. The mutation rates and eradication success rates were compared. RESULTS: The overall CAM-based eradication success rate was 84% (54/64): 62% (13/21) for CAM-resistant strains, and 95% (39/41) for CAM-sensitive strains. When the mutation rate of the 23S rRNA gene was 20% or lower for both positions (2142 and 2143), the eradication success rate was 90% or more. However, when the mutation rate was 20% or higher, the eradication success rate was lower (60%). CONCLUSIONS: The mutation rate of the CAM-resistance gene was related to the success of eradication therapy, as determined via pyrosequencing analysis.

Time-series transcriptome analysis of peripheral blood mononuclear cells obtained from individuals who received the SARS-CoV-2 mRNA vaccine.

Messenger ribonucleic acid (mRNA) vaccination against coronavirus disease 2019 (COVID-19) is an effective prevention strategy, despite a limited understanding of the molecular mechanisms underlying the host immune system and individual heterogeneity of the variable effects of mRNA vaccination. We assessed the time-series changes in the comprehensive gene expression profiles of 200 vaccinated healthcare workers by performing bulk transcriptome and bioinformatics analyses, including dimensionality reduction utilizing the uniform manifold approximation and projection (UMAP) technique. For these analyses, blood samples, including peripheral blood mononuclear cells (PBMCs), were collected from 214 vaccine recipients before vaccination (T1) and on Days 22 (T2, after second dose), 90, 180 (T3, before a booster dose), and 360 (T4, after a booster dose) after receiving the first dose of BNT162b2 vaccine (UMIN000043851). UMAP successfully visualized the main cluster of gene expression at each time point in PBMC samples (T1-T4). Through differentially expressed gene (DEG) analysis, we identified genes that showed fluctuating expression levels and gradual increases in expression levels from T1 to T4, as well as genes with increased expression levels at T4 alone. We also succeeded in dividing these cases into five types based on the changes in gene expression levels. High-throughput and temporal bulk RNA-based transcriptome analysis is a useful approach for inclusive, diverse, and cost-effective large-scale clinical studies.

Clinical outcomes and predictors of technical failure of endoscopic transpapillary gallbladder drainage in acute cholecystitis.

BACKGROUND AND AIMS: Endoscopic transpapillary gallbladder drainage (ETGBD) has been performed as an alternative therapy against cholecystectomy in patients with acute cholecystitis. To date, few studies have reported the safety, efficacy, and factors affecting ETGBD. We evaluated the clinical outcomes and predictors of technical failure of ETGBD. METHODS: Patients with acute cholecystitis who underwent ETGBD were retrospectively reviewed, and consecutive patients were included in the study. The technical success rate, clinical success rate, adverse events, and the predictors associated with the technical failure of ETGBD were investigated. RESULTS: A total of 242 patients were enrolled in the study. The technical success rate of ETGBD and clinical success rate of technically successful ETGBD cases were 87% and 93%, respectively. We experienced cystic duct injury in 24 patients as an ETGBD-related adverse event, and pancreatitis in 12 patients as an endoscopic retrograde cholangiopancreatography-related adverse event. Multivariate analysis indicated that cystic duct injury was the independent predictor associated with the technical failure of ETGBD (odds ratio, 11; 95% confidence interval, 3.9-29; p < 0.001). CONCLUSIONS: ETGBD was a safe and effective treatment method for acute cholecystitis with acceptable adverse events. There was no predictor based on the information from patient characteristics; however, cystic duct injury was associated with the technical failure of ETGBD.

Colonic TRPV4 overexpression is related to constipation severity.

BACKGROUND: Chronic constipation is prevalent and involves both colon sensitivity and various changes in intestinal bacteria, particularly mucosa-associated microflora. Here we examined regulatory mechanisms of TRPV4 expression by co-culturing colon epithelial cell lines with intestinal bacteria and their derivatives. We also investigated TRPV4 expression in colon epithelium from patients with constipation. METHODS: Colon epithelial cell lines were co-cultured with various enterobacteria (bacterial components and supernatant), folate, LPS, or short chain fatty acids. TRPV4 expression levels and promoter DNA methylation were assessed using pyrosequencing, and microarray network analysis. For human samples, correlation coefficients were calculated and multiple regression analyses were used to examine the association between clinical background, rectal TRPV4 expression level and mucosa-associated microbiota. RESULTS: Co-culture of CCD841 cells with P. acnes, C. perfringens, or S. aureus transiently decreased TRPV4 expression but did not induce methylation. Co-culture with clinical isolates and standard strains of K. oxytoca, E. faecalis, or E. coli increased TRPV4 expression in CCD841 cells, and TRPV4 and TNF-alpha expression were increased by E. coli culture supernatants but not bacterial components. Although folate, LPS, IL-6, TNF-alpha, or SCFAs alone did not alter TRPV4 expression, TRPV4 expression following exposure to E. coli culture supernatants was inhibited by butyrate or TNF-alphaR1 inhibitor and increased by p38 inhibitor. Microarray network analysis showed activation of TNF-alpha, cytokines, and NOD signaling. TRPV4 expression was higher in constipated patients from the terminal ileum to the colorectum, and multiple regression analyses showed that low stool frequency, frequency of defecation aids, and duration were associated with TRPV4 expression. Meanwhile, incomplete defecation, time required to defecate, and number of defecation failures per 24 h were associated with increased E. faecalis frequency. CONCLUSIONS: Colon epithelium cells had increased TRPV4 expression upon co-culture with K. oxytoca, E. faecalis, or E. coli supernatants, as well as TNFα-stimulated TNFαR1 expression via a pathway other than p38. Butyrate treatment suppressed this increase. Epithelial TRPV4 expression was increased in constipated patients, suggesting that TRPV4 together with increased frequency of E. faecalis may be involved in the pathogenesis of various constipation symptoms.

Clinical evaluation of a novel molecular diagnosis kit for detecting Helicobacter pylori and clarithromycin-resistant using intragastric fluid.

BACKGROUND: Although there are many Helicobacter pylori (H. pylori) diagnostic methods, the culture and antibiotic susceptibility test is an important method for selecting the most effective H. pylori eradication regimen. However, this diagnostic method is complicated and takes several days; therefore, the development of a rapid and simple diagnostic method is required. Eradication failure due to clarithromycin (CAM) resistance should also be considered. In this study, we report the clinical evaluation of point-of-care testing (POCT) kit using intragastric fluid, a novel kit for detecting H. pylori and CAM resistance. MATERIALS AND METHODS: The study participants were 143 patients suspected of H. pylori infection and had an endoscopic examination. The novel diagnostic kit diagnosed H. pylori infection and CAM resistance-associated mutation using intragastric fluid. To diagnose H. pylori infection, the relationship between the diagnostic kit and conventional diagnostic methods (urea breath test, stool antigen test, culture test, and real-time polymerase chain reaction [PCR]) was evaluated. For CAM resistance-associated mutation detection, the concordance between the diagnostic kit and antibiotic susceptibility test was evaluated. RESULTS: The diagnosis of H. pylori infection with the novel molecular diagnostic kit using intragastric fluid showed significant relationship with conventional diagnostic methods. Especially when the culture was control, the sensitivity was 100% (67/67), the specificity was 95.9% (71/74), and the overall concordance was 97.9% (138/141). The detection of CAM resistance-associated mutations had a concordance rate of 97.0% (65/67) when compared with the antibiotic susceptibility test. CONCLUSIONS: The H. pylori molecular POCT kit uses intragastric fluid as a sample and can diagnose H. pylori infection and detect CAM resistance-associated mutations within an hour. This novel kit is expected to prove useful in selecting the most effective eradication regimen for H. pylori.

Effects of endoscopic injection sclerotherapy for esophagogastric varices on portal hemodynamics and liver function.

OBJECTIVES: To identify patients suitable for endoscopic injection sclerotherapy (EIS) by evaluating their portal hemodynamics and liver function. METHODS: We selected 58 patients with esophagogastric varices (EGV) and liver cirrhosis (LC) related to either hepatitis C virus (C) (n = 19), hepatitis B virus (n = 2), alcohol (AL) (n = 20), C + AL (n = 6), non-alcoholic steatohepatitis (n = 6), others (n = 3), or non-LC (n = 2). All patients underwent EIS. We measured their portal venous tissue blood flow (PVTBF) and hepatic arterial tissue blood flow (HATBF) using xenon computed tomography before and after EIS. We classified them into increased group and decreased group according to the PVTBF to identify the predictors that contribute to PVTBF increase post-EIS. RESULTS: Low value of indocyanine green retention at 15 min (ICG-R15), the absence of paraesophageal veins, and low baseline PVTBF/HATBF (P/A) ratio predicted increased PVTBF in the multivariate logistic analysis (odds ratio (OR) 10.46, p = 0.0391; OR 12.45, p = 0.0088; OR 13.57, p = 0.0073). The protein synthetic ability improved 1 year post-EIS in increased group. Cox proportional hazards regression identified alcohol drinking (hazard ratio; 3.67, p = 0.0261) as an independent predictor of EGV recurrence. CONCLUSIONS: Patients with low ICG-R15, low P/A ratio, and the absence of paraesophageal veins were probable predictors of PVTBF improvement post-EIS. In addition, the improvement of hepatic hemodynamics likely enhanced liver function following EIS.

Combination of artificial intelligence-based endoscopy and miR148a methylation for gastric indefinite dysplasia diagnosis.

BACKGROUND AND AIM: Gastrointestinal endoscopy and biopsy-based pathological findings are needed to diagnose early gastric cancer. However, the information of biopsy specimen is limited because of the topical procedure; therefore, pathology doctors sometimes diagnose as gastric indefinite for dysplasia (GIN). METHODS: We compared the accuracy of physician-performed endoscopy (trainee, n = 3; specialists, n = 3), artificial intelligence (AI)-based endoscopy, and/or molecular markers (DNA methylation: BARHL2, MINT31, TET1, miR-148a, miR-124a-3, NKX6-1; mutations: TP53; and microsatellite instability) in diagnosing GIN lesions. We enrolled 24,388 patients who underwent endoscopy, and 71 patients were diagnosed with GIN lesions. Thirty-two cases of endoscopic submucosal dissection (ESD) in 71 GIN lesions and 32 endoscopically resected tissues were assessed by endoscopists, AI, and molecular markers to identify benign or malignant lesions. RESULTS: The board-certified endoscopic physicians group showed the highest accuracy in the receiver operative characteristic curve (area under the curve [AUC]: 0.931), followed by a combination of AI and miR148a DNA methylation (AUC: 0.825), and finally trainee endoscopists (AUC: 0.588). CONCLUSION: AI with miR148s DNA methylation-based diagnosis is a potential modality for diagnosing GIN.

Heterogeneity assessment of vaccine-induced effects using point-of-care surrogate neutralization test for severe acute respiratory syndrome coronavirus 2.

INTRODUCTION: Coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic even after vaccination. We aimed to identify immunological heterogeneity over time in vaccinated healthcare workers using neutralization antibodies and neutralizing activity tests. METHODS: Serum samples were collected from 214 healthcare workers before vaccination (pre) and on days 22, 90, and 180 after receiving the first dose of BNT162b2 vaccine (day 0). Neutralization antibody (NAb, SARS-CoV-2 S-RBD IgM/IgG) titers and two kinds of surrogate virus neutralization tests (sVNTs) were analyzed (UMIN000043851). RESULTS: The NAb (SARS-CoV-2 S-RBD IgG) titer peaked on day 90 after vaccination (30,808.0 µg/ml ± 35,211; p < 0.0001) and declined on day 180 (11,678.0 µg/ml ± 33,770.0; p < 0.0001). The neutralizing activity also peaked on day 90 and declined with larger individual differences than those of IgG titer on day 180 (88.9% ± 15.0%, 64.8% ± 23.7%, p < 0.0001). We also found that the results of POCT-sVNT (immunochromatography) were highly correlated with those of conventional sVNT (ELISA). CONCLUSIONS: Neutralizing activity is the gold standard for vaccine efficacy evaluation. Our results using conventional sVNT showed large individual differences in neutralizing activity reduction on day 180 (64.8% ± 23.7%), suggesting an association with the difference in vaccine efficacy. POCT-sVNT is rapid and user-friendly; it might be used for triage in homes, isolation facilities, and event venues without restrictions on the medical testing environment.

Incidence and management of cystic duct perforation during endoscopic transpapillary gallbladder drainage for acute cholecystitis.

BACKGROUND AND AIM: Evidence regarding the incidence and clinical outcome of cystic duct perforation (CDP) during endoscopic transpapillary gallbladder drainage (ETGBD) is inadequate. The present study aimed to evaluate the incidence and management of CDP during ETGBD. METHODS: Between March 2011 and December 2019, 249 patients underwent initial ETGBD for acute cholecystitis. The incidence of CDP was retrospectively examined and the outcomes between the CDP and non-CDP groups were compared. RESULTS: CDP during ETGBD occurred in 23 (9.2%) of 249 patients (caused by guidewire in 15 and cannula in 8). ETGBD was successful in 10 patients following CDP. In 13 patients who failed ETGBD, 11 underwent bile duct drainage during the same session; nine patients underwent gallbladder decompression by other methods, such as percutaneous drainage. Clinical resolution for acute cholecystitis was achieved in 20 patients, and no bile peritonitis was noted. ETGBD technical success rates (45.3% vs. 91.2%, p < 0.001), ETGBD procedure times (66.5 vs. 54.8 min, p = 0.041), and hospitalization periods (24.5 vs. 18.7 days, p = 0.028) were significantly inferior in the CDP group (n = 23) compared with the non-CDP group (n = 216). There were no differences in clinical success and adverse events other than CDP between both groups. CONCLUSIONS: Cystic duct perforation reduced the ETGBD technical success rate. However, even in patients with cystic duct perforation, an improvement of acute cholecystitis was achieved by subsequent successful ETGBD or additional procedures, such as percutaneous drainage.

Cancer-related genetic variants of Helicobacter pylori strains determined using gastric wash-based whole-genome analysis with single-molecule real-time technology.

Helicobacter pylori (H. pylori) are a primary factor in the pathogenesis of gastric cancer (GC); GC ranks third among cancer-related mortality. A clear understanding of the H. pylori genome factors underlying GC is necessary to develop more effective methods to prevent GC. A single-molecule real-time DNA sequencing-based H. pylori genome-wide association study analysis was performed using the H. pylori genome present in five early-stage GC (EGC) and five non-GC clinical DNA samples recovered from gastric washes. A total of 275 genes with 702 nucleotide variants (NVs) were found to be common to three or more patients with EGC but no non-GC patients (single-NV: 654/702, 93.2%; multi-NV: 40/702, 5.7%; deletion: 3/702, 0.4%; insertion: 3/702, 0.7%). Gene ontology analysis of H. pylori revealed that genes involved in the mitochondrial electron transport system, glycolytic processes and the TCA cycle were highly enriched. Cancer-related NVs were most frequently found in a member of the Helicobacter outer membrane protein family, hopL. In particular, one of the NVs in hopL was a novel six-nucleotide insertion (1159095̂1159096, TACTTC); this mutant was detected more frequently in a validation set of 50 additional EGC samples (22/50, 44.0%) than in 18 non-GC samples (3/18, 16.7%, P = .04). These results suggest that the hopL variant is associated with the development of GC and may serve as a genetic biomarker of H. pylori virulence and GC risk. Our assay can serve as a potent tool to expand our understanding of bacteria-associated tumorigenesis.

Tip-in endoscopic mucosal resection for large colorectal sessile polyps.

BACKGROUND: Tip-in endoscopic mucosal resection (EMR) is a modified EMR technique using which en bloc resection of large colorectal sessile polyps can be performed; however, its usefulness for colorectal sessile polyps of > 20 mm has not been reported. This study examined treatment outcomes of tip-in and conventional EMR for large colorectal sessile polyps of ≥ 20 mm. METHODS: This was a retrospective case-control study conducted at a single tertiary center in Japan. Subjects included those with large colorectal sessile polyps of ≥ 20 mm, excluding pedunculated-type polyps, who underwent endoscopic resection between January 2010 and January 2019. The primary outcome was endoscopic treatment outcomes when using tip-in and conventional EMR, and the secondary outcome was the local recurrence rate after endoscopic treatment. RESULTS: Forty-three colorectal lesions were treated using tip-in EMR and 83 using conventional EMR. Tip-in EMR had a significantly higher en bloc resection rate (90.7% vs. 69.8.%), and significantly shorter treatment duration (6.64 ± 0.64 min vs. 10.47 ± 0.81 min) than conventional EMR. However, for lesions > 30 mm, en bloc resection rate was 50.0% and 52.6% for tip-in and conventional EMR, respectively, indicating no significant difference. Perforation rates with tip-in and conventional EMR were 4.6% and 3.6%, respectively, indicating no significant difference. Local recurrence was examined in 80 cases who were followed up for > 6 months after endoscopic resection; recurrence rate was 0% and 7.0% in tip-in and conventional EMR cases, respectively, without significance difference. CONCLUSIONS: Tip-in EMR showed high en-block resection rate, particularly in polyps of < 30 mm, and no residual tumor was found. This technique is a potential endoscopic treatment alternative for large colorectal sessile polyps of ≥ 20 mm.

Evaluation of a new point-of-care quantitative reverse transcription polymerase chain test for detecting severe acute respiratory syndrome coronavirus 2.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is rapidly spreading worldwide, and the resultant disease, coronavirus disease (COVID-19), has become a global pandemic. Although there are multiple methods for detecting SARS-CoV-2, there are some issues with such tests, including long processing time, expense, low sensitivity, complexity, risk of contamination, and user friendly. This study evaluated the reproducibility and usability of a new point-of-care test (POCT) using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) for detecting SARS-CoV-2. METHODS: Samples from 96 patients with suspected SARS-CoV-2 infection were assessed using the real-time qRT-PCR-based POCT and the conventional real-time qRT-PCR method based on the Japanese National Institute of Infectious Diseases guidelines (registration number: jRCT1032200025). RESULTS: The real-time qRT-PCR-based POCT had a positive agreement rate of 90.0% (18/20), a negative agreement rate of 100% (76/76), and a total agreement rate of 97.9% (94/96), and the significantly high score of questionnaire survey (total score p < 0.0001). In the two cases in which real-time qRT-PCR-based POCT results did not match conventional real-time qRT-PCR test results, the SARS-CoV-2 RNA copy numbers were 8.0 copies per test in one case and below the detection limit in the other case when quantified using conventional real-time qRT-PCR. All patients could be triaged within 1 day using the real-time qRT-PCR-based POCT without invalid reports. CONCLUSIONS: The real-time qRT-PCR-based POCT not only had high reproducibility and useability but also allowed rapid patient triage. Therefore, it may be helpful in clinical settings.

A rare case of Epstein-Barr virus-positive mucocutaneous ulcer that developed into an intestinal obstruction: a case report.

BACKGROUND: Epstein-Barr virus-positive mucocutaneous ulcer (EBV-MCU) is a new category of mature B-cell neoplasms. Ulcers occur in the oropharyngeal mucosa, skin, and gastrointestinal tract. The onset of EBV-MCU is suggested to be related to the decreased immunity of the patient, the causes of which include the use of immunosuppressive agents and aging. EBV-MCU may regress spontaneously and it often has a benign course after the dose reduction or discontinuation of immunosuppressive agents or during follow-up. Here, we report the case of a patient who required surgical resection for the intestinal obstruction arising from EBV-MCU. CASE PRESENTATION: A Japanese elderly male visited our hospital with chief complaints of a palpable mass and dull pain in the left upper quadrant, loss of appetite, and weight loss. Although abdominal computed tomography and total colonoscopy (TCS) revealed a tumor with circumferential ulcer in the transverse colon, histopathological analysis of a biopsy specimen of this lesion showed only nonspecific inflammation. Because the tumor spontaneously regressed during the time he underwent tests to obtain a second opinion from another hospital, TCS was reperformed on the patient. TCS revealed that the tumor decreased in size and the inflammatory changes in the surrounding mucosa tended to improve; however, tightening of the surrounding mucosa due to scarring was observed. Another histopathological analysis of a biopsy specimen showed widespread erosion of the mucosa and the formation of granulation tissue with marked infiltration of various inflammatory cells into the mucosal tissue of the large intestine. Moreover, some of the B-lymphocyte antigen CD20-positive B cells were also positive for EBV-encoded small RNA-1, suggesting the possibility of EBV-MCU. Later, the tumor developed into an intestinal obstruction; thus, the transverse colon was resected. Histopathological analysis of the resected specimen demonstrated scattered Hodgkin and Reed-Sternberg-like multinucleated large B cells in addition to EBER-1-positive cells. The patient was finally diagnosed as having EBV-MCU. CONCLUSIONS: This is the first report of a case of EBV-MCU that developed into an intestinal obstruction requiring surgical resection. It is necessary to consider the possibility of EBV-MCU when examining an ulcerative or tumorous lesion in the gastrointestinal tract.

Pancreatic duct guidewire placement for biliary cannulation as a risk factor for stone residue after endoscopic transpapillary stone removal.

BACKGROUND: Recent improvements in stone extraction implements and apparatus have lessened the complexity of the endoscopic bile duct stone treatment. However, despite confirmation of complete removal, cases of residual stones have been reported, which can result in recurrent biliary symptoms, cholangitis, and pancreatitis and considerably increase cost given the need for repeat imaging and/or procedures. To date, risk factors for residual bile duct stones following endoscopic retrograde cholangiopancreatography (ERCP) extraction have not been thoroughly evaluated. This study retrospectively investigated the incidence and risk factors of residual bile duct stones following extraction via ERCP. METHODS: We retrospectively reviewed all ERCP cases that underwent endoscopic bile duct stone extraction between April 2014 and March 2019. A total of 505 patients were enrolled and evaluated for the incidence and risk factors of residual bile duct stones after ERCP. RESULTS: The rate of residual stones was 4.8% (24/505). Residual stones were detected by computed tomography (12/24) or magnetic resonance cholangiopancreatography (12/24). In univariate analyses, a large number of stones (P = 0.01), long procedure time (P = 0.005), and performance of the pancreatic duct guidewire placement method (P-GW) for selective bile duct cannulation (P = 0.01) were the factors involved in residual stones. In multiple logistic regression analysis, performing P-GW was retained as the only independent factor of residual stones (adjusted odds ratio, 3.44; 95% CI, 1.19-9.88; P = 0.02). CONCLUSIONS: When removing bile duct stones with a pancreatic guidewire in place, paying attention to residual stones is necessary.

Microsatellite instability in cancer: a novel landscape for diagnostic and therapeutic approach.

Defective DNA mismatch repair creates a strong mutator phenotype, recognized as microsatellite instability (MSI). Various next-generation sequencing-based methods for evaluating cancer MSI status have been established, and NGS-based studies have thoroughly described MSI-driven tumorigenesis. Accordingly, high-frequency MSI (MSI-H) has been detected in 81 tumor types, including those in which MSI was previously underrated. The findings have increased the use of immunotherapy, which is assumed to be efficient in tumors having a high mutation burden and/or neoantigen load. In MSI tumorigenesis, positively and negatively selected driver gene mutations have been characterized in colorectal cancers. Recent advancements in genome-wide studies of MSI-H cancers have developed novel diagnostic and therapeutic approaches, including CXCR2 inhibitor, a synthetic lethal therapy targeting the Werner gene and inhibition of nonsense-mediated mRNA decay. MSI is a predictive marker for chemotherapy as well as immunotherapy. Thus, analyses of MSI status and MSI-related alterations in cancers are clinically relevant. We present an update on MSI-driven tumorigenesis, focusing on a novel landscape of diagnostic and therapeutic approaches.

Mouthwash-Based Highly Sensitive Pyro-Genotyping for Nine Sexually Transmitted Human Papilloma Virus Genotypes.

Human papillomavirus (HPV) is a common sexually transmitted infection worldwide, which spreads via contact with infected genital, anal, and oral/pharyngeal areas (oral sex) owing to diverse manners of sexual intercourse. In this study, we devised an oral HPV detection method using mouthwash waste fluids that causes less psychological resistance to visiting the outpatient otolaryngology departments. We successfully detected only the specific unique reverse sequencing probe (using pyro-genotyping) and identified the nine genotypes of HPV targeted for vaccination by pyrosequencing the mouthwash waste fluids of non-head and neck cancer patient volunteers (n = 52). A relatively large number (11/52) of mouthwash waste fluids tested positive for HPV (21.2%; genotype 6, n = 1; 11, n = 1; 16, n = 1; and 18, n = 8). These results surpassed the sensitivity observed testing the same specimens using the conventional method (1/52, 1.9%). Our method (pyro-genotyping) was developed using nine HPV genotypes targeted for vaccination and the results were highly sensitive compared to those of the conventional method. This less expensive, high-throughput, and simple method can be used for detecting oral HPV infection with fewer socio-psychological barriers.

Diagnostic Ability of Endoscopic Bile Cytology Using a Newly Designed Biliary Scraper for Biliary Strictures.

BACKGROUND: A new device with metallic wires for scrape cytology was developed. AIMS: To compare the diagnostic performance of scrape cytology and conventional cytology during endoscopic retrograde cholangiopancreatography for biliary strictures. METHODS: A total of 420 cases with biliary stricture underwent transpapillary bile cytology. Among them, there are 79 cases with scrape cytology using the new device (scrape group) and 341 cases with conventional cytology (control group). Seventy-two and 174 cases underwent biliary biopsy at the same time as bile cytology in the scrape and control group, respectively. RESULTS: The sensitivity for malignancy of bile cytology in the scrape and control group was 41.2% [pancreatic cancer (PC): 23.1%, biliary cancer (BC): 52.5%] and 27.1% (PC: 16.3%, BC: 38.0%), respectively (P = 0.023). When analyzed PC and BC, respectively, there was no significant difference between the two groups. In the both groups, the sensitivity was significantly higher for BC than PC. In the scrape group, there was no difference in the sensitivity between cytology and biopsy [39.7% (PC: 17.4%, BC: 55.3%)], but in the control group, a significantly lower sensitivity was observed with cytology than biopsy (36.4% (PC: 19.7%, BC: 50.0%)) (P = 0.046). When analyzed PC and BC, respectively, there was no significant difference between cytology and biopsy. The sensitivity of combined cytology and biopsy was 55.6% (PC: 30.4%, BC: 71.1%) in the scrape group and 47.0% (PC: 24.6%, BC: 64.3%) in the control group. CONCLUSION: Scrape bile cytology for biliary strictures may be superior to conventional cytology.

Unique Biological Activity and Potential Role of Monomeric Laminin-γ2 as a Novel Biomarker for Hepatocellular Carcinoma: A Review.

Laminin (Ln)-332 consists of α3, ß3, and γ2 chains, which mediate epithelial cell adhesion to the basement membrane. Ln-γ2, a component of Ln-332, is frequently expressed as a monomer in the invasion front of several types of malignant tissues without simultaneous expression of Ln-α3 and/or Ln-ß3 chains. Moreover, monomeric Ln-γ2 induces tumor cell proliferation and migration in vitro. These unique biological activities indicate that monomeric Ln-γ2 could be a candidate biomarker for early cancer surveillance. However, the present immune method for monomeric Ln-γ2 detection can only predict its expression, since no antibody that specifically reacts with monomeric γ2, but not with heterotrimeric γ2 chain, is commercially available. We have, therefore, developed monoclonal antibodies to specifically detect monomeric Ln-γ2, and devised a highly sensitive method to measure serum monomeric Ln-γ2 levels using a fully automated chemiluminescent immunoassay (CLIA). We evaluated its diagnostic value in sera from patients with several digestive cancers, including hepatocellular carcinoma (HCC), and found serum monomeric Ln-γ2 to be a clinically available biomarker for HCC surveillance. The combination of monomeric Ln-γ2 and prothrombin induced by Vitamin K Absence II (PIVKA-II) may be more sensitive for clinical diagnosis of HCC than any currently used combination.

DNA methylation at hepatitis B viral integrants is associated with methylation at flanking human genomic sequences.

Integration of DNA viruses into the human genome plays an important role in various types of tumors, including hepatitis B virus (HBV)-related hepatocellular carcinoma. However, the molecular details and clinical impact of HBV integration on either human or HBV epigenomes are unknown. Here, we show that methylation of the integrated HBV DNA is related to the methylation status of the flanking human genome. We developed a next-generation sequencing-based method for structural methylation analysis of integrated viral genomes (denoted G-NaVI). This method is a novel approach that enables enrichment of viral fragments for sequencing using unique baits based on the sequence of the HBV genome. We detected integrated HBV sequences in the genome of the PLC/PRF/5 cell line and found variable levels of methylation within the integrated HBV genomes. Allele-specific methylation analysis revealed that the HBV genome often became significantly methylated when integrated into highly methylated host sites. After integration into unmethylated human genome regions such as promoters, however, the HBV DNA remains unmethylated and may eventually play an important role in tumorigenesis. The observed dynamic changes in DNA methylation of the host and viral genomes may functionally affect the biological behavior of HBV. These findings may impact public health given that millions of people worldwide are carriers of HBV. We also believe our assay will be a powerful tool to increase our understanding of the various types of DNA virus-associated tumorigenesis.

Efficacy of ledipasvir/sofosbuvir with or without ribavirin for 12 weeks in genotype 1b HCV patients previously treated with a nonstructural protein 5A inhibitor-containing regimen.

AIM: The therapeutic benefit of adding ribavirin (RBV) to 12 weeks of ledipasvir/sofosbuvir (LDV/SOF) for patients who experienced failure of a previous nonstructural protein (NS) 5A inhibitor-containing regimen is unclear. METHODS: A total of 29 genotype 1b HCV patients who had failed prior daclatasvir (DCV) plus asunaprevir (ASV) treatment were retreated for 12 weeks of LDV/SOF, with or without RBV. Antiviral efficacy and predictive factors associating with a sustained virological response at 24 weeks (SVR24) were evaluated retrospectively. RESULTS: SVR24 was achieved in 67% (10/15) of patients who received LDV/SOF with, and 64% (9/14) without, RBV. The SVR24 rates were 80% in patients with, and 58% without, mild fibrosis (FIB-4 < 3.25). The SVR24 rate was lower with unfavorable IL28B rs8099917 SNP genotypes; specifically, the TT, TG and GG had SVR24 rates of 78%, 50% and 40%. The SVR24 rate was lower with a poor response to prior DCV plus ASV, where relapse, viral breakthrough and no response had SVR24 rates 71%, 58% and 0%. The SVR24 rate was lower with the number of NS5A resistance-associated substitutions (RAS), where 2, 3, 4 and 5 RAS had SVR24 rates of 78%, 67%, 50% and 0%. A patient with an NS5A-P32 deletion, which shows resistance to next-generation NS5A inhibitors, was retreated with LDV/SOF with RBV and achieved SVR24. CONCLUSIONS: The addition of RBV to 12 weeks of LDV/SOF has little therapeutic benefit when retreating patients in whom a prior NS5A inhibitor-containing regimen had failed.