Comparative analysis of bile metabolic profile in patients with biliary obstruction complicated by Clonorchis sinensis infection.

Background: Clonorchiasis is an important foodborne parasitic disease. However, eggs of Clonorchis sinensis (C. sinensis) cannot be detected in feces during biliary obstruction. Moreover, many diseases can cause biliary obstruction, such as gallstones, adenocarcinoma, cholangiocarcinoma and Ascaris lumbricoides infection. Therefore, it is of great significance to distinguish between patients of biliary obstruction and biliary obstruction with C. sinensis infection. Methods: A total of 48 biliary obstruction patients were enrolled, including 23 infected with C. sinensis (C. sinensis) (OB+C.s) and 25 non-infected subjects (OB). The bile samples were collected by endoscopic retrograde cholangiopancreatography and analyzed using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF MS). Additionally, multivariate statistical analysis methods were employed to identify differential metabolites. Next, bile amino acid levels were determined by targeted metabolomics analysis. Result: A total of 146 and 132 significant metabolites were identified in electrospray ionization (ESI)+ and ESI- modes, respectively. The levels of amino acids (asparagine, glutamate, ornithine) and polyamines (spermidine and spermine) were significantly changed. Targeted analysis showed that the levels of amino acids (such as L-arginine, L-glutamine, L-lysine, L-propionic, and L-tyrosine) were lower in OB+C.s patients compared to those in OB patients. Marked metabolic pathways were involved in "Glutathione metabolism", "Caffeine metabolism", "Alanine, aspartate and glutamate metabolism", "Arginine and proline metabolism", "Purine metabolism", "Beta-Alanine metabolism", and "D-glutamine and D-glutamate metabolism". Conclusion: These results show that there were significant differences between OB+C.s and OB patients, especially in amino acids. The metabolic signature and perturbations in metabolic pathways may help to better distinguish OB+C.s and OB patients.

First description of Chloromyxum squali Gleeson and Adlard, 2012 (Myxozoa) in the Mediterranean Sea in a new host Squalus blainville (Chondrichthyes: Squalidae): morphological, ultrastructural and phylogenetic data.

Chloromyxum squali Gleeson and Adlard, 2012 originally described from Squalus acanthias Linnaeus, 1758 is reported for the first time from the gallbladder of Squalus blainville (Risso, 1827) caught from the eastern coast of Tunisia. In the present study, this chloromyxid was described based on morphological and ultrastructural features combined with molecular analysis of 18S rDNA. Young plasmodia were found attached to the gallbladder, while mature plasmodia and myxospores were observed floating free in the bile. Mature plasmodia were polysporic, subspherical in shape, measured 97.8 ± 0.5 µm long and 63.4 ± 0.4 µm wide. Mature myxospores were ovoid with a pointed anterior end, measuring 10.2 ± 0.5 µm long and 8.3 ± 0.5 µm wide. Two asymmetrical shell valves adhered together along an S-shaped suture line. Each valve has 5-7 elevated surface ridges parallel to suture line. A bundle of long caudal filaments extended from the basal end of shell valves. Four pyriform polar capsules equal in size, measuring 3.1 ± 0.4 µm long and 2.5 ± 0.3 µm wide, were situated at the same level in the anterior pole of the myxospore, each with a polar filament coiled in 7-8 turns. Pairwise comparisons among the SSU rDNA sequences revealed significant similarity between Chloromyxum squali infecting S. acanthias with the sequence obtained in this study. Phylogenetic analysis revealed that C. squali clustered in the clade of Chloromyxum species infecting the gallbladder of marine Chondrichthyes. Chloromyxum squali showed a seasonal variation of prevalence with significantly higher prevalence noted in summer and in autumn and absence of infection in winter.

Mast cell hyperplasia in Opisthorchis viverrini-associated cholecystitis.

Despite significant advances in understanding the role of the immune response in Opisthorchis viverrini-associated carcinogenesis, little is known about how infection induces gall bladder disease. This study investigated whether mast cells are activated in cholecystitis associated with O. viverrini, gall bladder specimens from ninety-two patients who had undergone cholecystectomy at the Khon Kaen Regional Hospital, Khon Kaen, Thailand. Two representative sections from the body of fresh gall bladder tissue were fixed in Carnoy's solution and embedded in paraffin wax. The paraffin sections were stained for mast cells and IgE plasma cells by the double histochemical and immunohistochemical method. The cells in the epithelium, lamina propria, muscular layer, and subserosa were counted and expressed as cells per square millimeter. The gall bladder bile was examined for the presence of O. viverrini eggs. Significantly higher mean mast cell numbers were found in the lamina propria (221.41 ± 16.01 vs 116.97 ± 14.61 cells per mm2; P < 0.005) of egg positive compared to egg negative groups, respectively. No comparable differences in mast cell number were observed in other layers. IgE plasma cells were rarely seen. The results suggest that mast cell hyperplasia occurs during cholecystitis in association with opisthorchiasis and may play a role in the pathogenesis of the disease.

Zschokkella epinepheli n. sp. (Myxosporea: Myxidiidae) infecting the gallbladder of the white grouper Epinephelus aeneus (Serranidae) from Tunisian waters.

A new coelozoic myxosporean species, Zschokkella epinepheli n. sp., collected from the gallbladder of the white grouper Epinephelus aeneus (Perciformes: Serranidae) from the bay of Bizerte, Tunisia, is described based on morphological and molecular characteristics. Myxospores and plasmodia were observed floating free in the bile. Mature plasmodia were polysporic and subspherical in shape, measuring 85.0-94.0 µm long and 70.0-82.0 µm wide. Mature myxospores were ovoid in valvular view, measuring 10.0 ± 1.7 (8.0-11.0) µm in length and 7.0 ± 0.3 (6.6-7.5) µm in width. Polar capsules were pyriform and equal in size, measuring 3.0 ± 0.2 (2.8-3.6) µm in length and 2.3 ± 0.3 (1.8-2.7) µm in width. Myxospore valves had 12-14 longitudinal striations. Based on the small subunit rDNA, the new species Z. epinepheli n. sp. differs from all other Zschokkella species for which there is a DNA sequence deposited in GenBank. Phylogenetic analysis revealed that Z. epinepheli n. sp. clustered in the marine subclade of Zschokkella species within the biliary tract IV clade. This is the first report of a Zschokkella species from the gallbladder of an epinephelin fishes.

Amino acids serve as an important energy source for adult flukes of Clonorchis sinensis.

Clonorchiasis, caused by chronic infection with Clonorchis sinensis (C. sinensis), is an important food-borne parasitic disease that seriously afflicts more than 35 million people globally, resulting in a socioeconomic burden in endemic regions. C. sinensis adults long-term inhabit the microaerobic and limited-glucose environment of the bile ducts. Energy metabolism plays a key role in facilitating the adaptation of adult flukes to crowded habitat and hostile environment. To understand energy source for adult flukes, we compared the component and content of free amino acids between C. sinensis-infected and uninfected bile. The results showed that the concentrations of free amino acids, including aspartic acid, serine, glycine, alanine, histidine, asparagine, threonine, lysine, hydroxylysine, and urea, were significantly higher in C. sinensis-infected bile than those in uninfected bile. Furthermore, exogenous amino acids could be utilized by adult flukes via the gluconeogenesis pathway regardless of the absence or presence of exogenous glucose, and the rate-limiting enzymes, such as C. sinensis glucose-6-phosphatase, fructose-1,6-bisphosphatase, phosphoenolpyruvate carboxykinase, and pyruvate carboxylase, exhibited high expression levels by quantitative real-time PCR analysis. Interestingly, no matter whether exogenous glucose was present, inhibition of gluconeogenesis reduced the glucose and glycogen levels as well as the viability and survival time of adult flukes. These results suggest that gluconeogenesis might play a vital role in energy metabolism of C. sinensis and exogenous amino acids probably serve as an important energy source that benefits the continued survival of adult flukes in the host. Our study will be a cornerstone for illuminating the biological characteristics of C. sinensis and the host-parasite interactions.

Clonorchiasis in Patients with Biliary and Pancreatic Diseases: Diagnosis and Risk Factors.

BACKGROUND: Many epidemiological studies have investigated the risk factors for clonorchiasis, but endoscopic findings of this disease in endoscopic retrograde cholangiopancreatography (ERCP) have not been well characterized. In this study, we evaluated clonorchiasis in ERCP in patients with biliary and pancreatic diseases. METHODS: This was a retrospective two-center study in hospitalized patients who received ERCP between January 2012 and October 2018. All patients were divided into clonorchiasis and nonclonorchiasis groups. Data were analyzed using univariate analysis and multivariate analyses. RESULTS: A total of 1119 patients were included, and clonorchiasis was diagnosed in 19.2% patients. Detection of Clonorchis sinensis eggs in bile samples was higher than that in fecal samples (85.9% vs. 58.7%; P = 0.001). In multivariate analysis, male patients (95% confidence interval (CI): 1.945-4.249, P = 0.001). In multivariate analysis, male patients (95% confidence interval (CI): 1.945-4.249, P = 0.001). In multivariate analysis, male patients (95% confidence interval (CI): 1.945-4.249, P = 0.001). In multivariate analysis, male patients (95% confidence interval (CI): 1.945-4.249, P = 0.001). In multivariate analysis, male patients (95% confidence interval (CI): 1.945-4.249, P = 0.001). In multivariate analysis, male patients (95% confidence interval (CI): 1.945-4.249. CONCLUSIONS: The detection of C. sinensis eggs was significantly higher in bile than in fecal samples; thus, bile samples represent a preferable sample for the diagnosis of clonorchiasis in patients with biliary obstruction. We found that male, age ≤ 60 years old, and CBD diameter < 12 mm were independent risk factors for clonorchiasis, while papilla fistula was a protective factor.C. sinensis eggs was significantly higher in bile than in fecal samples; thus, bile samples represent a preferable sample for the diagnosis of clonorchiasis in patients with biliary obstruction. We found that male, age ≤ 60 years old, and CBD diameter < 12 mm were independent risk factors for clonorchiasis, while papilla fistula was a protective factor.

Toxoplasma gondii-associated cholecystitis in a cat receiving immunosuppressive treatment.

A 9-year-old female neutered domestic shorthair cat diagnosed with immune-mediated thrombocytopenia that was treated with prednisolone and cyclosporine, was presented for anorexia, vomiting, increased liver enzymes, and hyperbilirubinemia. Abdominal ultrasound revealed a markedly thickened gallbladder and common bile duct wall. Bile cytology detected severe neutrophilic inflammation and protozoal zoites. Suspected Toxoplasma gondii infection was confirmed by real-time PCR of bile. The cat was treated with clindamycin and ursodeoxycholic acid for 6 weeks, recovered and remained stable for 2 years despite ongoing immunosuppressive treatment. Thereafter, the cat was presented with suspicion of intestinal lymphoma, and recurrence of toxoplasmosis was diagnosed. Following treatment with clindamycin and prednisolone over 4 weeks the cat was euthanized. This is the first report of Toxoplasma gondii zoites detected in bile fluid from a cat with cholecystitis. Pathogenesis of toxoplasmosis in cats is still not fully understood. Although immunosuppression can represent a relevant predisposing factor, other factors, such as virulence of the parasite and genetic polymorphism of the host, can also play an important role. Toxoplasmosis should be included as a differential diagnosis in cats developing clinical signs of an inflammatory disease while receiving immunosuppressive treatment.

A Clonorchiasis Case of a Leopard Cat, Prionailurus bengalensis euptilurus, Diagnosed by Ultrasonography and Egg Detection in Republic of Korea.

A clonorchiasis case in a captive leopard cat, Prionailurus bengalensis euptilurus, was confirmed by ultrasonographic findings and egg morphologies found in the bile juice sample in the Korea. The leopard cat was introduced from the wild habitat of Gyeongsangnam-do, to Cheongju Zoo in Cheongju-si, Chungcheongbuk-do, Korea in August 2014. Physical examinations were basically performed for quarantine and check-up health. The cat was comparatively good in health except anorexia. The cyst-like bile duct dilation and the increased echogenicity of gall bladder wall and hepatic parenchyma were observed by ultrasonography. Ultrasound-guided needle biopsy was conducted for collecting bile juice and the specimens were observed under light microscope. The numerous small trematode eggs were detected in the bile juice sample of the light microscopy. The eggs were 25-33 (28±3) µm by 18-22 (20±1) µm in size and showed typical characteristics of Clonorchis sinensis egg, i.e., a dominantly developed operculum, shoulder rim and dust-like wrinkles in surface. To treat the liver fluke infection, 20 mg/kg of praziquantel was orally administered only once to the case. Follow-up studies including fecal examinations were conducted during 2 years after treatment. But no more eggs were detected from the case. In the present study, we described the first clonorchiasis case of leopard cat, which was confirmed by ultrasonographic findings and egg morphologies from the bile juice sample in Korea.

A post-mortem study of bovine fasciolosis in the Mitidja (north center of Algeria): prevalence, risk factors, and comparison of diagnostic methods.

The present study was conducted to determine the prevalence of fasciolosis and investigate the associated risk factors (sex, age, and season) in slaughtered cattle of the Mitidja area. This survey also aimed to compare three diagnostic methods: liver inspection, microscopic bile examination, and ELISA test. Liver and bile of 1400 cattle were examined for the presence of adult and eggs of Fasciola hepatica, respectively. Based on the results obtained by liver and bile examination, a subset of 206 cattle from the 1400 was selected for antibodies detection using the ELISA test. Liver inspection and microscopic bile examination showed prevalences of 2.86% and 5.50%, respectively. The difference between the two methods was highly significant (p < 0.001). The overall prevalence of bovine fasciolosis found by both methods was 6.07%. The disease was more prevalent in females (11.96%) than in males (2.43%), and in older cattle (12.30%) than in younger ones (2.36%). The ELISA test showed some conflicting results: 23.61% of cattle previously diagnosed positive with liver inspection and bile examination were found seronegative, and 3.73% of cattle previously diagnosed negative were found to be seropositive. The sensitivity and specificity of microscopic bile examination were respectively 80% (CI 65.2-89.5%) and 96.7% (CI 95.6-97.5%) as compared with the liver inspection. Likewise, this same method has a sensitivity of 81.36% (CI 69.6-89.3%) and a specificity of 88.97% (CI 82.8-93.1%) as compared with the ELISA test. Our results show that microscopic bile examination is able to detect more infected cattle than liver inspection. In addition to being simple, fast, inexpensive, sensitive, and specific, it is an effective method for the diagnosis of fasciolosis.

Percutaneous Ultrasound-guided Cholecystocentesis and Bile Analysis for the Detection of Platynosomum spp.-Induced Cholangitis in Cats.

BACKGROUND: Examination of bile could be useful to diagnose Platynosomum spp.-induced cholangitis in cats. Obtaining bile via percutaneous ultrasound-guided cholecystocentesis (PUC) is possible but raises safety concerns in cats with severe cholecystitis. OBJECTIVES: The objectives of this study were to investigate the use of PUC to collect bile samples from cats with known platynosomosis and to determine if bile analysis could be a diagnostic test. ANIMALS: Twenty-seven free-roaming cats positive for Platynosomum spp. eggs via fecal examination. METHODS: In this prospective study, fecal egg counts were performed by double centrifugation with Sheather's solution. Bile was collected using PUC from anesthetized cats. Egg counts in bile were performed with a stereoscope. Euthanasia and postmortem examination were performed immediately after PUC. RESULTS: All cats had ultrasound (US) evidence of cholangitis or cholecystitis. Thirty-nine PUCs were performed with 14 cats having 2 PUCs 12 or 24 days apart. Postmortem examinations showed no overt gallbladder damage or leakage but fresh blood was noted in the gallbladder lumen of 3 cats. Median Platynosomum spp. egg counts were higher in bile (1450 eggs/mL; IQR, 400; 5138 eggs/mL) as compared to feces (46 eggs/mL; IQR, 10; 107 eggs/mL) (P < .001). CONCLUSION AND CLINICAL IMPORTANCE: Bile egg count analysis is an alternative method with higher egg counts as compared to fecal egg count analysis for the diagnosis of platynosomosis. Obtaining bile via US guidance is technically feasible and safe in cats with cholangitis/cholecystitis. Cholecystocentesis and bile analysis are especially relevant for those cats with chronic cholangitis/cholecystitis and negative fecal egg counts for Platynosomum.

Molecular Identification of Biliary Isospora Belli: A Case Report.

This report describes the novel sampling of bile from the biliary endoscopic intervention for the molecular identification of parasite infection.A 63-year-old Vietnamese man underwent travel health examination in our hospital. Physical examination showed that his height was 159 cm and weight was 41 kg. He had a 15-year history of intermittent abdominal pain and frequent episodes of diarrhea. Laboratory tests revealed raised eosinophil count (23%, normal range [NR] 0-5), absolute eosinophil count (1899/µL, NR 50-350), and levels of serum immunoglobulin E (3770 IU/mL, NR < 100), aspartate transaminase (270 U/L, NR 0-37), alanine transaminase (210 U/L, NR 0-40), and total bilirubin (1.8 mg/dL, NR 0.2-1.4); however, the serum alkaline phosphatase level was normal (65 U/L, NR 28-94) and non-reactive result for serum human insufficiency virus antibody.Magnetic resonance cholangiopancreatography revealed diffuse dilatation of the biliary tree; the common hepatic and pancreatic duct diameters increased to 1.86 cm and 0.61 cm, respectively.Endoscopic retrograde cholangiopancreatography was performed and a 10-Fr model plastic biliary stent was inserted and flushed with 20 cc normal saline; thereafter, the bile was collected and sent for DNA sequencing. Isospora belli (IB) infection was identified by a polymerase chain reaction.Trimethoprim-sulfamethoxazole 800 mg q6h was administered for 1 month. Liver enzyme levels normalized and negative for concentration method of ova study. The patient was doing well and weighed 51 kg at the outpatient clinic visit 3 months later.This bile sampling with molecular identification has not been described in the literature. We believe that an acute IB infection through fecal-oral transmission may progress to chronic infection of the hepatobiliary system, leading to biliary obstruction and jaundice.

Sarcocystid organisms found in bile from a dog with acute hepatitis: a case report and review of intestinal and hepatobiliary Sarcocystidae infections in dogs and cats.

Sarcocystidae is a family of coccidian protozoa from the phylum Apicomplexa that includes Toxoplasma, Neospora, Sarcocystis, Hammondia, and Besnoitia spp. All species undergo a 2-host sexual and asexual cycle. In the definitive host, replication is enteroepithelial, and infection is typically asymptomatic or less commonly causes mild diarrhea. Clinical disease is most frequently observed in the intermediate host, often as an aberrant infection, and is mostly associated with neurologic, muscular, or hepatic inflammation. Here, we review the literature regarding intestinal Sarcocystidae infections in dogs and cats, with emphasis on the life cycle stages and the available diagnostic assays and their limitations. We also report the diagnostic findings for an 11-year-old dog with acute neutrophilic hepatitis, biliary protozoa, and negative biliary culture. Although Toxoplasma and Neospora IgG titers were both high, PCR for these 2 organisms was negative for bile. The organisms were identified by 18S rDNA PCR as most consistent with Hammondia, either H heydorni or H triffittae. This is the first report of presumed Hammondia organisms being found in canine bile.

Reclassification of Eimeria pogonae Walden (2009) as Choleoeimeria pogonae comb. nov. (Apicomplexa: Eimeriidae).

The presented paper provides a reclassification of Eimeria pogonae from Pogona vitticeps into the correct genus Choleoeimeria. A description of exogenous and endogenous stages of biliary coccidium is given. Sporulation of the oocysts was endogenous. The mature oocysts contained four sporocysts each with two sporozoites. Oocysts were ellipsoidal in shape, with average length/width ratio 1.7 and measured 28.4 (SD1.5) × 16.8 (SD 1.5). The micropyle, residuum, and polar granules were absent from the sporulated oocysts. Ovoidal in shape, sporosysts without Steida bodies contained residuum and two elongated and boat-shaped sporozoites. The endogenous stages of the coccidia were located mainly in the epithelium of bile ducts; however, single-epithelium cells of the gallbladder were also infected.

Clonorchis sinensis ova in bile juice cytology from a patient with severe hyperbilirubinemia and portal vein thrombosis.

Infection with the trematode Clonorchis sinensis is the most common human fluke infection in East Asian populations. Although this infection is associated with obstructive jaundice or choledocholithiasis, portal vein thrombosis has not been reported. Here, we report the first case of a 60-year-old man who had both C. sinensis infection and portal vein thrombosis with severe hyperbilirubinemia (75.4 mg/dl). He initially presented with abdominal pain and jaundice. Computed tomography revealed gallstones, common bile duct calculus, and thrombus in the left main branch of the portal vein. A nasobiliary tube was inserted under endoscopic retrograde cholangiography. Cytology of the bile juice revealed many C. sinensis eggs. The abdominal pain and jaundice improved following choledocholithotomy and combination treatment with a chemotherapeutic agent and anti-coagulant. This case suggests that inflammation around the portal vein as a result of C. sinensis infection has the potential to evoke portal vein thrombosis. Such cases should be treated with both a chemotherapeutic agent and anti-coagulant therapy. In conclusion, the possibility of infection with C. sinensis should be considered in patients presenting with hyperbilirubinemia and portal vein thrombosis, particularly in East Asian populations.

In vivo assessment of closantel ovicidal activity in Fasciola hepatica eggs.

Anthelmintic resistance in livestock parasites is currently a worldwide problem. Fasciola hepatica is a cosmopolitan parasite which causes considerable loss in sheep and cattle production systems all over the world. Chemotherapy is currently the main tool available for its control. The intensive use of triclabendazole, the drug of choice for more than 20 years, has resulted in the development of resistant strains. The therapeutic options are adulticides such as closantel (salicylanilide anthelmintic that binds extensively to plasma albumin) to treat chronic fascioliasis in sheep, and cattle. In the present work, an Egg Hatch Assay (EHA) and morphometric studies were used to evaluate in vivo the ovicidal activity and morphology F. hepatica eggs, recovered from closantel treated sheep collected at different time intervals post treatment. Statistically significant differences (p < 0.0001) were observed in egg morphometry between the control and the treated groups in all the parameters studied. Eggs recovered from treated animals tend to be narrower and longer. Significant differences were found in the embryonation and hatching of eggs between 36 h post treatment (32, 5%) vs. approximately 85% in control, 12 h and 24 h post treatment. Our results confirm that closantel affects in vivo the normal development of the eggs. As one of the first effects, this drug affects the performance of the trematode's reproductive physiology. Even though closantel treated animals may still eliminate eggs in the first days post treatment, these are not viable.

UV irradiation responses in Giardia intestinalis.

The response to ultraviolet light (UV) radiation, a natural stressor to the intestinal protozoan parasite Giardia intestinalis, was studied to deepen the understanding of how the surrounding environment affects the parasite during transmission. UV radiation at 10 mJ/cm(2) kills Giardia cysts effectively whereas trophozoites and encysting parasites can recover from UV treatment at 100 mJ/cm(2) and 50 mJ/cm(2) respectively. Staining for phosphorylated histone H2A showed that UV treatment induces double-stranded DNA breaks and flow cytometry analyses revealed that UV treatment of trophozoites induces DNA replication arrest. Active DNA replication coupled to DNA repair could be an explanation to why UV light does not kill trophozoites and encysting cells as efficiently as the non-replicating cysts. We also examined UV-induced gene expression responses in both trophozoites and cysts using RNA sequencing (RNA seq). UV radiation induces small overall changes in gene expression in Giardia but cysts show a stronger response than trophozoites. Heat shock proteins, kinesins and Nek kinases are up-regulated, whereas alpha-giardins and histones are down-regulated in UV treated trophozoites. Expression of variable surface proteins (VSPs) is changed in both trophozoites and cysts. Our data show that Giardia cysts have limited ability to repair UV-induced damage and this may have implications for drinking- and waste-water treatment when setting criteria for the use of UV disinfection to ensure safe water.

In vitro maintenance of Clonorchis sinensis adult worms.

Clonorchis sinensis is a biological carcinogen inducing human cholangiocarcinoma, and clonorchiasis is one of the important endemic infectious diseases in East Asia. The present study investigated survival longevity of C. sinensis adult worms in various in vitro conditions to find the best way of keeping the worms longer. The worms were maintained in 0.85% NaCl, 1×PBS, 1×Locke's solution, RPMI-1640, DMEM, and IMDM media, and in 1×Locke's solution with different supplements. All of the worms died within 3 and 7 days in 0.85% NaCl and 1×PBS, respectively, but survived up to 57 days in 1×Locke's solution. The worms lived for 106 days in DMEM, and 114 days in both RPMI-1640 and IMDM media. The survival rate in RPMI-1640 medium was the highest (50%) compared to that in DMEM (20±10%) and in IMDM (33.3±25.2%) after 3 months. The 1×Locke's solution with 0.005% bovine bile supplement showed increased duration of maximum survival from 42 days to 70 days. Higher concentration of bile supplements than 0.005% or addition of glucose were disadvantageous for the worm survival. The worms died rapidly in solutions containing L-aspartic acid, L-glutamic acid, and adenine compared to L-arginine, L-serine, and L-tryptophan. In conclusion, the 1×Locke's solution best supports the worms alive among inorganic solutions for 57 days, and the RPMI-1640 medium maintains living C. sinensis adults better and longer up to 114 days in vitro than other media.

[Detection of Clonorchis sinensis eggs in the ground gallbladder stones by microscopy].

Sera, feces, bile and gallbladder stones were collected from 179 patients who accepted gallbladder-preserving cholelithotomy during the period of January to June 2010 at the general surgery department in the Second People's Hospital of Panyu District in Guangzhou. Rapid colloidal gold immunochromatography was used to detect IgG against Clonorchis sinensis. C. sinensis eggs were examined by fecal direct smear, and in bile sediments and ground gallbladder stones. The results showed that the positive rate of rapid colloidal gold immunochromatographic assay for IgG was 51.4%, and the egg positive rate in feces, bile sediments and gallbladder stones was 30.7%, 44.7% and 69.8%, respectively. The detection rate of fecal direct smear was the lowest, while that of the gallbladder stone examination was the highest (P < 0.05). Those patients with egg-positive feces and/or bile sediments were all with egg-positive gallbladder stones.

Development of a real-time PCR assay for the detection of Clonorchis sinensis DNA in gallbladder bile and stone samples from patients with cholecystolithiasis.

High prevalence of cholecystolithiasis in parts of East Asia has been postulated to be associated with Clonorchis sinensis infection. This study describes the development of a TaqMan-based real-time PCR assay for the detection of C. sinensis DNA in gallbladder bile and stone samples from patients with cholecystolithiasis. Primers and probe targeting the cytochrome c oxidase subunit 1 gene of mitochondrial DNA proved to be highly specific for C. sinensis and did not amplify other related heterogeneous DNA samples. The detection limit of this assay was 0.1 pg of adult C. sinensis genomic DNA. All of the egg-positive samples determined by microscopy yielded positive results by real-time PCR assay and that genetic testing of gallbladder stones using real-time PCR was considered as the most effective means for assessing C. sinensis infection status. This assay not only contributes to a greater understanding of stone pathogenesis but also benefits patients with cholecystolithiasis by facilitating effective diagnosis, treatment, and relapse prevention.