Bile Samples Research Articles

Biliary Metabolome Profiling for Evaluation of Liver Metabolism and Biliary Tract Function Related to Organ Preservation Method and Degree of Ischemia in a Porcine Model.

The development of surgical techniques, immunosuppressive strategies and new organ preservation methods have meant that transplant centers have to face the problem of an insufficient number of organs for transplantation concerning the constantly growing demand. Therefore, using organs from expanded criteria donors and developing new analytical solutions to find parameters or compounds that would allow a more efficient assessment of organ quality before transplantation are options for meeting this challenge. This study proposed bile metabolomic analysis to evaluate liver metabolism and biliary tract function depending on the organ preservation method and degree of warm ischemia time. The analyses were performed on solid-phase microextraction-prepared bile samples from porcine model donors with mild (heart beating donor [HBD]) and moderate warm ischemia (donation after circulatory death [DCD]) grafts subjected to static cold storage (SCS) or normothermic ex vivo liver perfusion (NEVLP) before transplantation. Bile produced in the SCS-preserved livers was characterized by increased levels of metabolites such as chenodeoxycholic acid, arachidonic acid and 5S-hydroxyeicosatetraeonic acid, as well as saturated and monounsaturated lysophosphatidylcholines (LPC). Such changes may be associated with differences in the bile acid synthesis pathways and organ inflammation. Moreover, it has been shown that NEVLP reduced the negative effect of ischemia on organ function. A linear relationship was observed between levels of lipids from the LPC group and the time of organ ischemia. This study identified metabolites worth considering as potential markers of changes occurring in preserved grafts.

Environmental chemicals and endogenous metabolites in bile of USA and Norway patients with primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is a complex bile duct disorder. Its etiology is incompletely understood, but environmental chemicals likely contribute to risk. Patients with PSC have an altered bile metabolome, which may be influenced by environmental chemicals. This novel study utilized state-of-the-art high-resolution mass spectrometry (HRMS) with bile samples to provide the first characterization of environmental chemicals and metabolomics (collectively, the exposome) in PSC patients located in the United States of America (USA) (n = 24) and Norway (n = 30). First, environmental chemical- and metabolome-wide association studies were conducted to assess geographic-based similarities and differences in the bile of PSC patients. Nine environmental chemicals (false discovery rate, FDR < 0.20) and 3143 metabolic features (FDR < 0.05) differed by site. Next, pathway analysis was performed to identify metabolomic pathways that were similarly and differentially enriched by the site. Fifteen pathways were differentially enriched (P < .05) in the categories of amino acid, glycan, carbohydrate, energy, and vitamin/cofactor metabolism. Finally, chemicals and pathways were integrated to derive exposure-effect correlation networks by site. These networks demonstrate the shared and differential chemical-metabolome associations by site and highlight important pathways that are likely relevant to PSC. The USA patients demonstrated higher environmental chemical bile content and increased associations between chemicals and metabolic pathways than those in Norway. Polychlorinated biphenyl (PCB)-118 and PCB-101 were identified as chemicals of interest for additional investigation in PSC given broad associations with metabolomic pathways in both the USA and Norway patients. Associated pathways include glycan degradation pathways, which play a key role in microbiome regulation and thus may be implicated in PSC pathophysiology.

Biliary giardiasis in raccoons (Procyon lotor)

Giardiasisis one of the most significant gastrointestinal diseases caused by a cosmopolitan protozoa Giardia duodenalis. The infection occurs in many animals, including wild raccoons (Procyonlotor), that may play a role in disseminating Giardia cysts into the environment and transmitting it to humans and domestic animals. Giardiasis has various clinical manifestations, including diarrhea, abdominal cramps, and upset stomach or nausea. Active trophozoites normally attach to the mucous membrane of the duodenum and upper jejunum, however few cases of biliarygiardiasis have been reported in humans. The aim of this study was to analyze the occurrence of Giardia localization in the gall bladder of raccoons. Bile and feces samples collected from each of the 34 test animals were used for the study. Giardia specific antigen was detected by a rapid immunochromatographic test using stool and bile samples. The overall prevalence was 38.2% (13/34, 95% CI, 21.0-55.4%). Positive results were obtained from 8 stool samples 23.5% (95% CI, 8.5-38.6%) and 13 bile samples 38.2% (95% CI, 21.0-55.4%). In all stool-positive raccoons, the infestation was confirmed by bile examination. In five cases a positive result was obtained only from the bile sample. The detection of the specific antigen GSA65 in the bile of the studied raccoons confirms that, similarly to humans, also in animals in the course of Giardia infection trophozoites can be localized both in the intestine and in the bile ducts.

Molecular diagnosis of pancreatobiliary tract cancer by detecting mutations and methylation changes in bile samples.

Patients with pancreatobiliary tract cancer usually have a poor clinical outcome, with a 5-year overall survival rate below 20%. This is mainly associated with the late diagnosis. In addition, the standard-of-care for patients with malignant biliary stenosis involves a major surgery, the Whipple procedure. An accurate preoperative diagnosis, including differentiation from benign diseases, is critical to avoid unnecessary treatment. Here we developed BileScreen, a sensitive detection modality for the diagnosis of pancreatobiliary tract cancer based on massively parallel sequencing mutation and methylation changes in bile samples. A total of 338 patients, from five hospitals in China, with pancreatobiliary system disorders were enrolled in this study between November 2018 and October 2020, and 259 were included for the analysis of BileScreen. We profiled 23 gene mutations and 44 genes with methylation modifications in parallel from bile samples, and set up a model for the detection of malignancy based on multi-level biomarkers. We applied the BileScreen assay in a training cohort (n=104) to set up the model and algorithm. The model was further evaluated in a validation cohort (n=105), resulting in 92% sensitivity and 98% specificity. The performance of BileScreen was further assessed in a prospective test cohort (n=50) of patients diagnosed with suspicious or negative pathology by endoscopic retrograde cholangiopancreatography and were confirmed in follow-up. BileScreen yielded 90% sensitivity and 80% specificity, and outcompeted serum carbohydrate antigen 19-9 in detecting pancreatobiliary tract cancer in all three cohorts, especially in terms of specificity. Taken together, BileScreen has the ability to interrogate mutations and methylation changes in bile samples in parallel, thus rendering it a potentially sensitive detection method to help in the diagnosis of pancreatobiliary tract cancer in a safe, convenient and less-invasive manner. This study was supported by the Capital's Funds for Health Improvement and Research (2020-2-4025 to S.H.), the National Natural Science Foundation of China (81972311 to H.Z.), CAMS Innovation Fund for Medical Sciences (CIFMS) (2017-12M-4-002 to H.Z.), the CAMS Innovation Fund for Medical Sciences(CIFMS) (2021-I2M-1-066 to CJQ), the Non-profit Central Research Institution Fund of Chinese Academy of Medical Sciences (2019PT310026 to H.Z.) and Sanming Project of Medicine in Shenzhen (SZSM202011010 to H.Z.).

Effect of various dietary fructose concentrations on the gallstone formation process in mice.

Background: little information is availaible on the effect of fructose on bile lipids. The first stage in the formation of gallstones corresponds to biliary cholesterol crystallization, derived from the vesicular transporters. The aim of this study was to investigate the influence of consuming diets with different fructose concentrations on serum lipids and their implications on gallstones formation. Methods: BALB/c mice divided into a control group as well as groups were treated with different fructose concentrations (10 %, 30 %, 50 % or 70 %) for different periods (1, 2 or 5 months). Blood, liver and bile samples were obtained. In bile samples, cholesterol and phospholipids levels were analyzed, and cholesterol transporters (vesicles and micelles) were separated by gel filtration chromatography. Results: treated animals showed: 1) increases in body weight similar to the control group; 2) a significant increase in plasma triglycerides only at very high fructose concentrations; 3) a significant increase in total serum cholesterol in the treatment for 1 month; 4) no variations in HDL-cholesterol; 5) a significant increase in serum glucose only at very high fructose concentrations in the second month of treatment; 6) no differences in the plasma alanine-aminotransferase activity; 7) a significant increase in liver triglyceride levels only at very high fructose concentrations; 8) no change in biliary lipid concentrations or in micellar and vesicular phospholipids. Conclusion: changes in plasma, liver and bile lipids were only observed at very high fructose concentrations diets. We conclude that fructose apparently does not alter the gallstone formation process in our experimental model.

Bacteriobilia frequency in patients afflicted by biliary obstruction at a tertiary care center

Background: Bile typically remains sterile within individuals possessing a normal biliary tract, but bacterial colonization arises with biliary obstruction. Infections often stem from ascending duodenal infection or bacterial translocation from the portal vein. Elevated common bile duct pressure due to obstruction, combined with infected bile, can lead to bacterial backflow into lymphatics, fostering cholangitis. In this study, 30 sequential bile samples were examined, scrutinizing the occurrence of bacteriobilia and comparing its incidence between benign and malignant etiologies. Aims and Objectives: Bacteriobilia typically manifests without symptoms. This study aimed to assess the prevalence of bacteriobilia in patients with obstructive jaundice undergoing endoscopic retrograde cholangiopancreatography (ERCP) at our institution, investigating associated risk factors. Furthermore, the study aimed to elucidate the microbiological profile of bile in obstructed biliary systems, make a comparative analysis of bacteriobilia frequency between benign and malignant obstructions, and delve into the drug sensitivity patterns of bacteria cultured from bile at our center. Materials and Methods: In a prospective, observational, and diagnostic study conducted at the Department of Digestive Health and Diseases, Government Peripheral Hospital, Anna Nagar, Chennai, from September 2011 to February 2012, with ethical committee approval and informed consent from all participants, we investigated 30 consecutive patients who underwent ERCP for biliary obstruction. The study cohort comprised 13 males and 17 females. Following successful biliary cannulation, bile samples were collected and subjected to microbiological analysis. Results revealed positive bile cultures in 16 patients, with 10 exhibiting benign etiology and 6 malignant. Gram-negative organisms, predominantly Klebsiella, were identified, displaying favorable susceptibility to cephalosporins while demonstrating resistance to ciprofloxacin. Results: Bile cultures yielded positive results in 16 patients, with 10 cases attributed to benign etiology and 6 to malignant etiology. The cultured organisms were predominantly Gram-negative, with Klebsiella exhibiting significant growth. Notably, these organisms exhibited favorable susceptibility to cephalosporins while displaying resistance to ciprofloxacin. Conclusion: Bile culture serves as a valuable tool for prompt diagnosis and pre-emptive management of complications. Gram-negative bacilli infections prevail within obstructed biliary systems. This study underscores the pivotal role of routine bile sampling during ERCP in obstructed biliary systems, offering predictive insights to avert complications like cholangitis.

Real-Time Fluorescence Imaging to Identify Cholangiocarcinoma in the Extrahepatic Biliary Tree Using an Enzyme-Activatable Probe

Introduction: Complete resection is the only possible treatment for cholangiocarcinoma in the extrahepatic biliary tree (eCCA), although current imaging modalities are limited in their ability to accurately diagnose longitudinal spread. We aimed to develop fluorescence imaging techniques for real-time identification of eCCA using an enzyme-activatable probe, which emits fluorescence immediately after activation by a cancer-specific enzyme. Methods: Using lysates and small tissue fragments collected from surgically resected specimens, we selected the most specific probe for eCCA from among 800 enzyme-activatable probes. The selected probe was directly sprayed onto resected specimens and fluorescence images were acquired; these images were evaluated for diagnostic accuracy. We also comprehensively searched for enzymes that could activate the probe, then compared their expression levels in cancer and non-cancer tissues. Results: Analyses of 19 samples (four cancer lysates, seven non-cancer lysates, and eight bile samples) and 54 tissue fragments (13 cancer tissues and 41 non-cancer tissues) revealed that PM-2MeSiR was the most specific fluorophore for eCCA. Fluorescence images of 7 patients were obtained; these images enabled rapid identification of cancerous regions, which closely matched histopathology findings in 4 patients. Puromycin-sensitive aminopeptidase was identified as the enzyme that might activate the probe, and its expression was upregulated in eCCA. Conclusion: Fluorescence imaging with PM-2MeSiR, which may be activated by puromycin-sensitive aminopeptidase, yielded generally high accuracy. This technique may be useful for real-time identification of the spread of eCCA during surgery and endoscopic examinations.

Feasibility of Raman spectroscopic identification of gall bladder cancer using extracellular vesicles extracted from bile.

Extracellular vesicles (EVs), which are heterogeneous membrane-based vesicles with bilayer cell membrane structures, could be versatile biomarkers for the identification of diverse diseases including cancers. With this potential, this study has attempted the Raman spectroscopic identification of gall bladder (GB) cancer by directly measuring the EV solution extracted from human bile without further sample drying. For this purpose, bile samples were obtained from four normal individuals and 21 GB polyp, eight hepatocellular carcinoma (HCC), and five GB cancer patients, and EVs were extracted from each of the bile samples. The Raman peak shapes of the EVs extracted from the GB cancer samples, especially the relative intensities of peaks in the 1560-1340 cm-1 range, were dissimilar to those of the samples from the normal, GB polyp, and HCC groups. The intensity ratios of peaks at 1537 and 1453 cm-1 and at 1395 and 1359 cm-1 of the GB cancer samples were lower and higher, respectively, than those of the samples of the remaining three groups. The differences of peak intensity ratios were statistically significant based on the Mann-Whitney U test. DNA/RNA bases, amino acids, and bile salts contributed to the spectra of EVs, and their relative abundances seemed to vary according to the occurrence of GB cancer. The varied metabolite compositions and/or structures of EVs were successfully demonstrated by the dissimilar peak intensity ratios in the Raman spectra, thereby enabling the discrimination of GB cancer.

Comparison of bacterial profile of gallbladder with gallstones from patients undergoing cholecystectomy due to complicated and uncomplicated cholelithiasis: changes in the epidemiological scenario.

cholelithiasis is a highly prevalent disease of the digestive system in the world. In Brazil, it is a routine condition, whose studies suggest a prevalence of around 10% of adults. Colonization of bile and gallstone pathogens can occur when there is bacterial stasis and proliferation. This proliferation is facilitated by the adhesion and biofilm formation capacity of some bacteria. There are also lithogenic processes that involve bacterial participation. Studies have shown changes in the microbiota of the gallbladder of patients undergoing cholecystectomy, which may impact empirical treatment with antibiotics. microbiological analyzes of the sonication fluid of the gallstones and of two samples with bile were performed. Identification and antimicrobial susceptibility testing were performed according to a standard routine. of the 34 patients, 76.4% were female. The age group was 48 years +/- 16.61. Acute cholecystitis occurred in 50% of cases. Bactobilia was evidenced in 32.1% of the cases. Klebisiella pneumoniae was noted as the most prevalent pathogen in acute cholecystitis; and Enterobacter sp, in cases of uncomplicated cholelithiasis. Greater sensitivity was obtained in the search for microorganisms in the sonication fluid samples of the stones in relation to the bile samples (p=0.0058). there was a higher prevalence of bactobilia in patients with acute cholecystitis compared to those with uncomplicated cholelithiasis. The use of sonication in bacterial investigation proved to be superior to the conventional method and can be considered.

Assessing hydroxylated polycyclic aromatic hydrocarbon (OHPAH) metabolites in bile of English sole (Parophrys vetulus) from Puget Sound, WA, USA by liquid chromatography/tandem mass spectrometry (LC-MS/MS)

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants that are widely monitored in marine biota from urbanized areas, due to their toxicity to aquatic organisms. Teleost fish can quickly metabolize PAHs into hydroxylated forms (OHPAHs) that, in some cases, are more toxic than the parent (unmetabolized) PAHs. But due to this fast metabolism, monitoring traditional parent PAHs in fish can cause underestimation on assessing PAH exposure. In addition, environmental levels of individual OHPAH metabolites are lacking in the literature worldwide. Therefore, we developed a rapid and accurate analytical method in which a number of individual OHPAHs metabolites are measured simultaneously in fish bile, via liquid chromatography coupled with tandem mass spectrometry, including low and high molecular weight mono- and diol-OHPAHs. We analyzed bile samples of 119 English sole (Parophrys vetulus) collected from 14 Puget Sound, WA, USA, sites, which has multiple sources of PAHs, including urban stormwater runoff, wastewater effluents, as well as an inactive creosote facility. The mean (± SD) biliary summed OHPAH (∑OHPAH) concentrations determined in English sole from urban, near-urban, and non-urban sites were 790 ± 1400 (n = 46), 310 ± 330 (n = 44) and 130 ± 200 (n = 29) ng/mL, respectively, with a maximum reaching 9400 ng/mL in a sample from an urban site. We compared these novel biliary OHPAH metabolite data with parent PAHs measured in stomach content of the same individual sole. Biliary ∑OHPAH concentrations were significantly correlated with the levels of ∑PAH in stomach content, however, with major differences in their distribution. We also demonstrated that biliary OHPAH metabolite data in English sole can potentially be used to distinguish different sampling sites due to a specific variety and intensity of PAH sources in the aquatic environment, which makes this a very important analytical approach for assessing PAH exposure in the environment.

ABO-Incompatible Liver Transplantation under the Desensitization Protocol with Rituximab: Effect on Biliary Microbiota and Metabolites.

Background: ABO-incompatible liver transplantation (ABOi LT) under the desensitization protocol with rituximab had excellent survival outcomes comparable to those of ABO-compatible liver transplantation (ABOc LT). In this work, we explored the effect of ABOi LT on recipients from the perspective of biliary microbiota and metabonomics. Methods: Liver transplant (LT) recipients treated at our center were enrolled in the study. In total, 6 ABOi LT recipients and 12 ABOc LT recipients were enrolled, and we collected their bile five times (during LT and at 2 days, 1 week, 2 weeks and 1 month after LT). The collected samples were used for 16S ribosomal RNA sequencing and liquid chromatography mass spectrometry analysis. Results: We obtained 90 bile samples. Whether in group ABOi LT or ABOc LT, the most common phyla in all of the samples were Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria. The most common genera were Lactobacillus, Weissella, Klebsiella, Pantoea and Lactococcus. There was no significant difference in the diversity between the two groups at 1 week, 2 weeks and 1 month after LT. However, the biggest disparities between the ABOi LT recipients and ABOc LT recipients were observed 2 days after LT, including increased biodiversity with a higher ACE, Chao1, OBS and Shannon index (p < 0.05), and more Staphylococcus in ABOi LT and binary−Jaccard dissimilarity, which indicated varying β-diversity (p = 0.046). These differences were not observed at 1 week, 2 weeks and 1 month after LT. The principal coordinate analysis (PCoA) revealed that the composition of the bile microbiota did not change significantly within 1 month after LT by longitudinal comparison. In an analysis of the bile components, the metabolites were not significantly different every time. However, four enrichment KEGG pathways were observed among the groups. Conclusion: These findings suggest that ABOi LT under the desensitization protocol with rituximab did not significantly affect the biliary microbiota and metabolites of recipients.

MICROBIOLOGICAL PATTERN IN BILIARY TRACT INFECTION AT CHO RAY HOSPITAL IN 2021

Background: MBiliary tract infection is a common cause of bacteremia with high morbidity and mortality, particularly in older patients with co-morbid disease or when there is a delay in diagnosis and treatment. This study is undertaken to determine the microbiological pattern and antibiotic resistance in biliary tract infection at Cho Ray Hospital in 2021.Subbjects and methods: NA retrospective study was conducted from January 2021 to Demcember 2021 at Cho Ray hospital. Bile samples were cultured on Blood agar, Mac-conkey agar. Bacterial strains were identified by the MALDI-TOF MS system and made susceptibility testing on the VITEK 2-compact system of Biomerieux. The antibiotic susceptibility were determined by mininum inhibitory concentration(MIC) according to CLSI.Results: Out of 595 bile samples sent for aerobic culture and susceptibility testing, 203 (34.1%) weresterile and 392 (65.9%) were found to be culture positive. Of the bile culture positives, 129 (21.7%)were polymicrobial. The most prevalent bacteria isolates were Escherichia coli 33.8% (102), Klebsiella pneumoniae 22.2% (67), Enterococcus spp. 16.6% (50), Pseudomonas aeruginosa 7.9% (24). The ESBL rate of Escherichia coli was 42.2%, while this rate of Klebsiella pneumoniae was 9%. The vancomycin resistant rate of Enterococcus spp. was 8.3%. In Pseudomonas aeruginosa, among beta-lactams, imipenem had the highest resistant rate (45.8%). No resistance to colistin had been reported.Conclusion: In biliary tract infections, about one-third cases had negative-culture results and about onefifth cases had polymicrobial infection. The common pathogens in biliary tract infections were Escherichia coli, Klebsiella pneumoniae, Enterococcus spp. and Pseudomonas aeruginosa. The resistance rate to commonly used antibiotics in treatment was quite high.

From the Editor’s Desk…

From the Editor’s Desk…

Antibiogram Analysis of Salmonella paratyphi A Isolated from Gall Bladder Patients in District Peshawar, Pakistan

Salmonella paratyphi A harbors gall bladder in the human body. It serves as a site of persistence for Salmonella paratyphi A. It is an enteric pathogen which has become resistant to many drugs. Therefore, the current study was designed for the identification and antibiogram analysis of S. paratyphi A, isolated from the gall bladder patients undergone cholecystectomy. It included 250 samples of bile, stone, and tissue of patients. The sampleswere cultured on blood, macConkey, and Salmonella Shigella media. Further identification was carried out by morphological oxidase test and Analytical Profile Index (API) strips, followed by antibiogram analysis of the isolates. In the current study, twenty-eight (11.2%) paratyphi A were isolated including 10 (10%) from male patients and 18 (12%) from female patients. Furthermore, 96 samples were found to be positive formiscellaneous growth including 53 with S. typhi (21.2%), 13 with Escherichia coli (5.2%), 09 with Klebsiella (3.6%), 07 with Providencia (2.8%), 05 with Pseudomonas (2%), 03 with Proteus (1.2%), and 06 with Staphylococcus aureus (2.4%). The distribution and susceptibility pattern of S. paratyphi A isolates was checked in different types of clinical specimens including bile, stones, tissue, bile/stones, bile/tissue, stones/tissue, and bile/stone/tissue. S. paratyphi A was distributed as follows: bile (11), stones (5), tissue (3), bile/ stones (4), stones/ tissue (1), bile/tissue (1), and bile/stones/tissue (3). The results of the antibiogram analysisfound that the isolates of Paratyphi A were resistant to sulfamethoxazole 23 (82.14%), cefixime 23 (82.14%),ceftriaxone (rocephin) 20 (71.42%), augmentin 19 (67.85%), and azithromycin 18 (64.28%). The increased susceptibility of these isolates was towards meronem 28 (100%), imipenem 28 (100%), cefoperazon + sulbactam (sulzone) 25 (89.28%), and amikacin 23 (82.14%). The current study signifies the use of the Antibiogram Analysis of Salmonella paratyphi A most susceptible and effective antibiotic options for gall bladder diseases complicated by S. paratyphi A, which showed resistance to ceftriaxone (rocephin), cefixime, sulfamethoxazole, azithromycin, and augmentin, while sensitivity to meropenem, imipenem, cefoperazone + sulbactam (sulzone), and amikacin. It makes the latter a better choice for treatment against the gall stone disease complicated with S. paratyphi A infection.

Role of bacteriocholia in development of infectious complications of pancreaticoduodenal resection

The incidence of infectious complications after pancreaticoduodenal resection remains significant. High frequency of bacteriocholia in this category of patients is a result of preoperative biliary drainage. The role of bacteriocholia in the development of surgical site infection and the methods of exposure to it are yet to be studied. The authors presented the analysis of Russian and foreign literature and considered the role of bacteriocholia in the development of surgical site infection after pancreaticoduodenal resection. Bacteriocholia appears to be one of the most significant factors in the surgical site infection after this kind of resection. Bacterial cultures in the intraoperative bile sample and the surgical microbiome are represented by pathogens resistant to standard drugs used for perioperative antibiotic prophylaxis. In order to decrease the incidence of surgical site infections after the pancreaticoduodenal resection it is essential to continue searching for the methods of exposure to bacteriocholia including revision of the approaches to perioperative administration of antibacterial agents.

Bile-volatile organic compounds in the diagnostics of pancreatic cancer and biliary obstruction: A prospective proof-of-concept study.

Detection of volatile organic compounds (VOCs) from bodily fluids with field asymmetric waveform ion mobility spectrometry (FAIMS) and related methods has been studied in various settings. Preliminary results suggest that it is possible to detect prostate, colorectal, ovarian and pancreatic cancer from urine samples. In this study, our primary aim was to differentiate pancreatic cancer from pancreatitis and benign tumours of the pancreas by using bile samples obtained during endoscopic retrograde cholangiopancreatography (ERCP). Secondarily, we aimed to differentiate all pancreatic region malignancies from all other kinds of benign causes of biliary obstruction. A bile sample was successfully aspirated from 94 patients during ERCP in Tampere University Hospital. Hospital and patient records were prospectively followed up for at least two years after ERCP. Bile samples were analysed using a Lonestar chemical analyser (Owlstone, UK) using an ATLAS sampling system and a split-flow box. Diagnoses and corresponding data from the analyses were matched and divided into two subcategories for comparison. Statistical analysis was performed using linear discriminant analysis, support vector machines, and 5-fold cross-validation. Pancreatic cancers (n=8) were differentiated from benign pancreatic lesions (n=9) with a sensitivity of 100%, specificity of 77.8%, and correct rate of 88%. All pancreatic region cancers (n=19) were differentiated from all other kinds of benign causes of biliary obstruction (n=75) with corresponding values of 21.1%, 94.7%, and 80.7%. The sample size was too small to try to differentiate pancreatic cancers from adjacent cancers. Analysing bile VOCs using FAIMS shows promising capability in detecting pancreatic cancer and other cancers in the pancreatic area.

A strategy to explore the quality markers of Ziziphi Spinosae semen by combining metabolic in vivo study with network pharmacology.

Ziziphi Spinosae semen (ZSS), the dried and ripe seed of Ziziphus jujube Mill. var. spinosa (Bunge) Hu ex H. F. Chou, has been used as a sedative in China and other Asian countries for over a millennium. However, its quality markers (Q-markers) are not completely clear. In this study, Q-markers selected by a metabolic in vivo study combined with network pharmacology are proposed for ZSS quality control. An UHPLC (ultra-high-performance liquid chromatography)-Q-Orbitrap-MS method was developed to identify or tentatively assign 48 components including 21 flavonoid C-glycosides, 2 flavonoid O-glycosides, 11 dammarane triterpenoid saponins, 13 alkaloids, and 1 other, using a diagnostic product ion filtering strategy in ZSS. Subsequently, 147 metabolites detected from serum, urine, bile, and feces samples of para-chlorophenylalanine-induced insomnia rats treated with ZSS aqueous extracts could be linked to their respective parent compounds, including 27 prototypes. Meanwhile, three metabolic networks of flavonoids, saponins, and alkaloids are preliminarily established and potential metabolic pathways are investigated under the insomnia condition. Finally, 12 key bioactive components against insomnia including magnoflorine, caaverine, coclaurine, norisocorydine, genkwanin, juzinrine, apigenin, jujubogenin, kaempferol-3-O-rutinoside, jujuboside A, jujuboside B, and spinosin with the highest degree values in component-target-pathways network were selected as Q-markers for the quality control of ZSS.

Campylobacter bilis, the second novel Campylobacter species isolated from chickens with Spotty Liver Disease, can cause the disease

Spotty Liver Disease (SLD) is a significant disease of commercial layer hens. It can cause up to 10 % flock mortalities and reduce egg production by 25 %. Campylobacter hepaticus has been identified as the main cause of the disease, although it also appears that predisposing factors, such as some form of stress, may increase the likelihood of clinical disease occurring. Recently, a newly identified species, Campylobacter bilis, was isolated from bile samples of clinical SLD affected chickens. To investigate the pathogenic potential of C. bilis two independent isolates were used in infection trials of layer hens. Within 6 days of oral challenge birds developed typical SLD liver lesions, demonstrating that both strains induced SLD. C. bilis could be recovered from all the challenged birds that developed SLD. Thus, each of the steps in Koch’s postulates have been fulfilled, confirming that C. bilis is an additional cause of SLD. A PCR method was developed which can specifically detect C. bilis from samples with complex microbiota. The identification of this newly discovered Campylobacter species as a second cause of SLD and the provision of a rapid method to detect the SLD causing bacterium will help with SLD vaccine development and epidemiology, thus assisting in the control of this important disease of poultry.

Unravelling Bile Viromes of Free-Range Laying Chickens Clinically Diagnosed with Spotty Liver Disease: Emergence of Many Novel Chaphamaparvoviruses into Multiple Lineages.

Spotty liver disease (SLD) causes substantial egg production losses and chicken mortality; therefore, it is a disease that concerns Australian egg farmers. Over the last few decades, much research has been conducted to determine the etiologic agents of SLD and to develop potential therapeutics; however, SLD still remains a major issue for the chicken industries globally and remained without the elucidation of potentially multiple pathogens involved. To help fill this gap, this study was aimed at understanding the viral diversity of bile samples from which the SLD-causing bacterium, Campylobacter hepaticus, has been isolated and characterised. The collected samples were processed and sequenced using high-throughput next-generation sequencing. Remarkably, this study found 15 galliform chaphamaparvoviruses (GaChPVs), of which 14 are novel under the genus Chaphamaparvovirus. Among them, nine were complete genomes that showed between 41.7% and 78.3% genome-wide pairwise similarities to one another. Subsequent phylogenetic analysis using the NS1 gene exhibited a multiple incursion of chaphamaparvovirus lineages, including a novel lineage of unknown ancestral history in free-range laying chickens in Australia. This is the first evidence of circulating many parvoviruses in chickens in Australia, which has increased our knowledge of the pathogen diversity that may have an association with SLD in chickens.

Detection of bile acids in small volume human bile samples via an amino metal-organic framework composite based solid-phase microextraction probe

In recent years, bile acids (BAs), the important component of bile, were found closely related to the occurrence and development of diseases, therefore, determination of BAs in bile samples is of great significance. However, biological matrix complexity and low concentrations of BAs were still challenging for BA detection in small amount of bile samples. In this work, a core-shell NH2-MIL101@mSiO2 was designed to improve the capture ability of BAs in biological samples, as well as possess good biocompatibility. Subsequently, solid-phase microextraction (SPME) probe of the NH2-MIL101@mSiO2 was coupled with HPLC-MS/MS to establish the analysis method for detecting eight BAs in bile samples. The established method received extraction efficiencies of (30–2143)-fold higher than those of the commercial probes and low limit of detection (LOD ≤ 0.21 ng mL−1). The miniaturization of SPME sampling devices, as well as the low LOD of this work, endowed this method advantage of low consumption of bile samples (30 μL). Based on the proposed method, eight BAs in bile samples of pancreatic cancer patients and cholelithiasis patients were detected successfully. A distinct difference was found in the concentrations of four targeted BAs in bile samples from pancreatic cancer patients and cholelithiasis patients. This work provided a method for quantification of eight BAs in small volume human bile samples, and it could open up a perspective regarding the relationship between BA metabolism and the occurrence of diseases.