New challenges in cholangiocarcinoma candidates for elective surgery: harnessing the microbiome dysbiosis.

BACKGROUND: The gut microbiota, composed by several species of microorganisms, works to preserve the liver-gut homeostasis and plays an important role during digestion and absorption of nutrients, and in the immune response of the host. In this review, we analyzed the influence of microbiota in patients with cholangiocarcinoma (CCA) who were candidates for elective surgery. METHODS: A literature review was conducted to identify papers that provided empiric evidence to support that the altered microbiota composition (dysbiosis) is related also to CCA development. RESULTS: Bacteria such as Helicobacter pylori, Helicobacter hepaticus, and Opisthorchis viverrini increase the risk of CCA. The most abundant genera were Enterococcus, Streptococcus, Bacteroides, Klebsiella, and Pyramidobacter in CCA's biliary microbiota. Additionally, levels of Bacteroides, Geobacillus, Meiothermus, and Anoxybacillus genera were significantly higher. An enrichment of Bifidobacteriaceae, Enterobacteriaceae, and Enterococcaceae families has also been observed in CCA tumor tissue. Microbiota is related to postoperative outcomes in abdominal surgery. The combination of caloric restriction diets in liver cancer or CCA increases the effect of the chemotherapy treatment. CONCLUSION: The correct use of nutrition for microbiota modulation according to each patient's needs could be a therapeutic tool in combination with elective surgery and chemotherapy to diminish side effects and improve prognosis. Further investigations are needed to fully understand the mechanisms by which they are related.

A comparative study of Helicobacter pylori infection in hamsters experimentally infected with liver flukes Opisthorchis felineus, Opisthorchis viverrini, or Clonorchis sinensis.

Helicobacter pylori causes a wide range of human diseases including cancer. Carcinogenic foodborne trematodes Opisthorchis viverrini, Clonorchis sinensis, and O. felineus might promote transmission and spread of H. pylori infection in the definitive mammalian host, which in turn might contribute to the liver fluke-associated malignancy. Our objectives were to find out whether liver flukes O. felineus, O. viverrini, and C. sinensis are carriers of Helicobacter pylori and to determine whether H. pylori is present in feces, bile, and stomach samples from the experimentally infected hamsters. We found that liver flukes are not reservoirs of H. pylori. Nevertheless, the prevalence of H. pylori and the H. pylori ureA gene copy number were significantly elevated after the infection. Overall, although the liver flukes O. felineus, C. sinensis, and O. viverrini are not reservoirs of H. pylori, the infection with the liver flukes significantly modifies the biliary and gut microbiota by increasing H. pylori abundance. This may be a feature of any liver fluke pathogenesis that have not previously been taken into account. Our findings appear to be novel in terms of comparative assessment of the host microbiota and Helicobacter abundance during epidemiologically important liver fluke infections.

The impact of Opisthorchis felineus infection and praziquantel treatment on the intestinal microbiota in children.

The presence of some species of helminths is associated with changes in host microbiota composition and diversity, which varies widely depending on the infecting helminth species and other factors. We conducted a prospective case-control study to evaluate the gut microbiota in children with Opisthorchis felineus infection (n=50) before and after anthelmintic treatment and in uninfected children (n=49) in the endemic region. A total of 99 children and adolescents aged between 7 and 18 years were enrolled to the study. Helminth infection was assessed before and at 3 months after treatment with praziquantel. A complex examination for each participant was performed in the study, including an assessment of the clinical symptoms and an intestinal microbiota survey by 16S rRNA gene sequencing of stool samples. There was no change in alpha diversity between O. felineus-infected and control groups. We found significant changes in the abundances of bacterial taxa at different taxonomic levels between the infected and uninfected individuals. Enterobacteriaceae family was more abundant in infected participants compared to uninfected children. On the genus level, O. felineus-infected participants' microbiota showed higher levels of Lachnospira, Escherichia-Shigella, Bacteroides, Eubacterium eligens group, Ruminiclostridium 6, Barnesiella, Oscillibacter, Faecalitalea and Anaerosporobacter and reduction of Blautia, Lachnospiraceae FCS020 and Eubacterium hallii group in comparison with the uninfected individuals. Following praziquantel therapy, there were significant differences in abundances of some microorganisms, including an increase of Faecalibacterium and decrease of Megasphaera, Roseburia. Enterobacteriaceae and Escherichia abundances were decreased up to the control group values. Our results highlight the importance of the host-parasite-microbiota interactions for the community health in the endemic regions.

Opisthorchiasis and the Microbiome.

The liver flukes Opisthorchis viverrini, O. felineus, and Clonorchis sinensis are closely related fish-borne trematodes endemic in East Asia, Eurasia, and Siberia. Following ingestion, the parasites locate to the biliary tree, where chronic infection frequently leads to cholangiocarcinoma (CCA). Infection with C. sinensis or O. viverrini is classified as a Group 1 carcinogen by the International Agency for Research on Cancer. Infection with O. felineus may also be carcinogenic. The mechanism(s) by which infection with these liver flukes culminates in CCA remain elusive, although they are likely to be multi-factorial. Not yet well studied is the influence of opisthorchiasis on the microbiome of the host despite reports that helminth parasites are capable of affecting the microbiome, potentially modulating gastrointestinal inflammation in response to the appearance of pathogenic strains of bacteria. Here, we review recent findings related to opisthorchiasis and the microbiome and related issues. In the hamster, a tractable model of infection with liver fluke and of infection-induced biliary morbidity and CCA, infection with O. viverrini perturbs the microbiome of the gastrointestinal tract, including increasing numbers of Lachnospiraceae, Ruminococcaceae, Lactobacillaceae, and others, while decreasing Porphyromonadaceae, Erysipelotrichaceae, and Eubacteriaceae. In addition, a complex microbial community associates with the parasites within the biliary tree, including Helicobacter pylori and related bacteria. Moreover, higher rates of infection with Helicobacter occur in Thailand in persons with opisthorchiasis in a liver fluke infection intensity-dependent manner. Experimental infection of hamsters with Opisthorchis felineus results in increased alpha diversity of the microbiota diversity in the biliary tract. In humans, infection with O. felineus modifies the composition of the biliary microbiome, with increasing numbers of species of Klebsiella, Aggregatibacter, Lactobacillus, Treponema, and others. Several phylotypes of Archaea occurred solely in bile from persons infected with O. felineus.

Opisthorchiasis-Induced Cholangiocarcinoma: How Innate Immunity May Cause Cancer.

Innate, inflammatory responses towards persistent Opisthorchis viverrini (OV) infection are likely to contribute to the development of cholangiocarcinoma (CCA), a liver cancer that is rare in the West but prevalent in Greater Mekong Subregion countries in Southeast Asia. Infection results in the infiltration of innate immune cells into the bile ducts and subsequent activation of inflammatory immune responses that fail to clear OV but instead may damage local tissues within the bile ducts. Not all patients infected with OV develop CCA, and so tumourigenesis may be dependent on multiple factors including the magnitude of the inflammatory response that is activated in infected individuals. The purpose of this review is to summarize how innate immune responses may promote tumourigenesis following OV infection and if such responses can be used to predict CCA onset in OV-infected individuals. It also hypothesizes on the role that Helicobacterspp., which are associated with liver fluke infections, may play in activation of the innate the immune system to promote tissue damage and persistent inflammation leading to CCA.

Elevated prevalence of Helicobacter species and virulence factors in opisthorchiasis and associated hepatobiliary disease.

Recent reports suggest that Opisthorchis viverrini serves as a reservoir of Helicobacter and implicate Helicobacter in pathogenesis of opisthorchiasis-associated cholangiocarcinoma (CCA). Here, 553 age-sex matched cases and controls, 293 and 260 positive and negative for liver fluke O. viverrini eggs, of residents in Northeastern Thailand were investigated for associations among infection with liver fluke, Helicobacter and hepatobiliary fibrosis. The prevalence of H. pylori infection was higher in O. viverrini-infected than uninfected participants. H. pylori bacterial load correlated positively with intensity of O. viverrini infection, and participants with opisthorchiasis exhibited higher frequency of virulent cagA-positive H. pylori than those free of fluke infection. Genotyping of cagA from feces of both infected and uninfected participants revealed that the AB genotype accounted for 78% and Western type 22%. Participants infected with O. viverrini exhibited higher prevalence of typical Western type (EPIYA ABC) and variant AB'C type (EPIYT B) CagA. Multivariate analyses among H. pylori virulence genes and severity of hepatobiliary disease revealed positive correlations between biliary periductal fibrosis during opisthorchiasis and CagA and CagA with CagA multimerization (CM) sequence-positive H. pylori. These findings support the hypothesis that H. pylori contributes to the pathogenesis of chronic opisthorchiasis and specifically to opisthorchiasis-associated CCA.

Coinfection with Helicobacter pylori and Opisthorchis viverrini Enhances the Severity of Hepatobiliary Abnormalities in Hamsters.

Persistent infection with Opisthorchis viverrini causes hepatobiliary abnormalities, predisposing infected individuals to cholangiocarcinoma (CCA). In addition, Helicobacter pylori is highly prevalent in most countries and is a possible risk factor for CCA; however, its role in enhancing hepatobiliary abnormality is unclear. Here, we investigated the effects of coinfection with H. pylori and O. viverrini on hepatobiliary abnormality. Hamsters were divided into four groups: (i) normal, (ii) H. pylori infected (HP), (iii) O. viverrini infected (OV), and (iv) O. viverrini and H. pylori infected (OV+HP). At 6 months postinfection, PCR and immunohistochemistry were used to test for the presence of H. pylori in the stomach, gallbladder, and liver. In the liver, H. pylori was detected in the following order: OV+HP, 5 of 8 (62.5%); HP, 2 of 5 (40%); OV, 2 of 8 (25%). H. pylori was not detected in normal (control) liver tissues. Coinfection induced the most severe hepatobiliary abnormalities, including periductal fibrosis, cholangitis, and bile duct hyperplasia, leading to a significantly decreased survival rate of experimental animals. The greatest thickness of periductal fibrosis was associated with a significant increase in fibrogenesis markers (expression of alpha smooth muscle actin and transforming growth factor beta). Quantitative reverse transcription-PCR revealed that the highest expression levels of genes for proinflammatory cytokines (interleukin-1 [IL-1], IL-6, and tumor necrosis factor alpha) were also observed in the OV+HP group. These results suggest that coinfection with H. pylori and O. viverrini increased the severity of hepatobiliary abnormalities to a greater extent than either single infection did.

Chronic Opisthorchis viverrini Infection Changes the Liver Microbiome and Promotes Helicobacter Growth.

Adults of Opisthorchis viverrini reside in the biliary system, inducing inflammation of bile ducts and cholangitis, leading to hepatobiliary disease (HBD) including cholangiocarcinoma. O. viverrini infection also has major implications for the bacterial community in bile ducts and liver. To investigate this in chronic O. viverrini infection (≥ 8 months p.i.), bacterial genomic DNA from livers of hamsters and from worms was investigated using culture techniques, PCR for Helicobacter spp. and high-throughput next-generation sequencing targeting the V3-V4 hypervariable regions of prokaryotic 16S rRNA gene. Of a total of 855,046 DNA sequence reads, 417,953 were useable after filtering. Metagenomic analyses assigned these to 93 operational taxonomic units (OTUs) consisting of 80 OTUs of bacteria, including 6 phyla and 42 genera. In the chronic O. viverrini-infected group, bacterial community composition and diversity were significantly increased compared to controls. Sequences of Fusobacterium spp. were the most common (13.81%), followed by Streptococcus luteciae (10.76%), Escherichia coli (10.18%), and Bifidobacterium spp. (0.58%). In addition, Helicobacter pylori (0.17% of sequences) was also identified in the liver of chronic O. viverrini infections, but not in normal liver. The presence of H. pylori was confirmed by PCR and by use of an antibody against bacterial antigen, supporting the metagenomics data. The identities of bacteria cultured for enrichment suggested that chronic O. viverrini infection changes the liver microbiome and promotes Helicobacter spp. growth. There may be synergy between O. viverrini and the liver microbiome in enhancing immune response-mediated hepatobiliary diseases.

Immunization and challenge shown by hamsters infected with Opisthorchis viverrini following exposure to gamma-irradiated metacercariae of this carcinogenic liver fluke.

Here we report findings to optimize and standardize conditions to attenuate metacercariae of Opisthorchis viverrini by ionizing radiation to elicit protective immune responses to challenge infection. Metacercariae were gamma-irradiated and the ability of irradiated metacercariae to prevent patent infection of challenge metacercariae in hamsters was determined, as well as their ability to induce a host antibody response. Metacercariae irradiated in a dose-dependent manner, with 3, 5, 10, 12, 20, 25 and 50 Gray, were used to infect Syrian golden hamsters by stomach gavage to ascertain the effect of irradiation on ability of the worms to establish infection. In addition, other hamsters were infected with metacercariae irradiated with 20-50 Gray, followed by challenge with intact/wild-type (non-irradiated) metacercariae to determine the protective effect as established by the numbers of adult flukes, eggs of O. viverrini in hamster faeces and anti-O. viverrini antibody titres. Significantly fewer worms were recovered from hamsters immunized with metacercariae irradiated at 20, 25 and 50 Gray than from control hamsters infected with intact metacercariae or 0 Gray, and the worms showed damaged reproductive organs. Faecal egg numbers were decreased significantly in hamsters immunized with 25 and 50 Gray metacercariae of O. viverrini. Moreover, hamsters administered metacercariae that were protected elicited a robust, specific anti-fluke immunoglobulin G response compared to control hamsters, suggesting a role for antibody in protection elicited by radiation-attenuated metacercariae.

Infection with the carcinogenic liver fluke Opisthorchis viverrini modifies intestinal and biliary microbiome.

Opisthorchis viverrini is a fish-borne trematode endemic in East Asia. Following ingestion, the flukes locate to the biliary tre where chronic infection frequently leads to cholangiocarcinoma (CCA). The mechanisms by which O. viverrini infection culminates in CCA remain unknown. An unexplored aspect is its influence on the host microbiome. In the hamster, infection with this pathogen reliably leads to CCA. Genomic DNAs of microbiota from colorectal contents and bile of hamsters and from whole O. viverrini were examined in this model of fluke-induced CCA. Microbial communities were characterized by high-throughput sequencing of variable regions 7-9 of prokaryotic 16S ribosomal DNA. Of ∼1 million sequences, 536,009 with useable reads were assignable to 29,776 operational taxonomy units (OTUs) and, in turn, to 20 phyla and 273 genera of Bacteria or Archaea. Microbial community analyses revealed that fluke infection perturbed the gastrointestinal tract microbiome, increasing Lachnospiraceae, Ruminococcaceae, and Lactobacillaceae, while decreasing Porphyromonadaceae, Erysipelotrichaceae, and Eubacteriaceae (P≤0.05). More than 60 OTUs were detected in the biliary system, which confirmed bacteriobilia and a noteworthy community of microbes associated with the parasites. The fluke-associated microorganisms included potential pathogens from the Enterobacteriaceae and Listeriaceae and others, including Cyanobacteria and Deinococci, usually found in external environments. Given that opisthorchiasis is distinguished from other helminth infections by a robust inflammatory phenotype with conspicuously elevated IL-6, and that inflammation of the biliary system leads to periductal fibrosis, which is a precursor of CCA, the flukes and their microbiota may together drive this distinctive immune response.

[The pathomorphosis of opisthorchiasis]. / Patomorfoz opistorkhoza.

Based on the data available in the literature and their own findings, the authors have come to the conclusion that the clinical picture of opisthorchiasis has undergone profound changes in the past decades, which may be regarded as a manifestation of induced and reduced pathomorphism. Uninduced pathomorphism appears as decreased invasion rates and as acute forms. Induced pathomorphism shows up in reduced invasion or by timely liquidation of invasion. The etiological and pathogenetic role of Helicobacter pylori in the development of the gastroduodenal pathology in patients with opisthorchiasis is considered as a manifestation of false pathomorphism.

[Characteristics of the immunity in typhoid infection associated with opisthorchiasis]. / Osobennosti immuniteta pri briushnotifoznoi infektsii, assotsiirovannoi s opistorkhozom.

Typhoid infection developing in persons with opisthorchiasis is characterized by the appearance of pronounced systemic immunity, that ensures a more favorable clinical course of this infection and promotes a decrease in the occurrence of diarrheal phenomena and bacteremia. At the same time, in typhoid patients, simultaneously affected by opisthorchiasis, a more intensive release of the infective agent into the environment is observed. This seemingly demonstrates the presence of disturbances in the local protective mechanisms regulating the process of the release of bacteria on the level of the gastrointestinal tract.

[Pathogenetic and immunological characteristics of different forms of the Salmonella typhi carrier state]. / Patogeneticheskaia i immunologicheskaia kharakteristika razlichnykh form briushnotifoznogo bakterionositel'stva.

Infectious granulomas with macrophages containing Salmonella typhi have been detected in the immune organs of the intestine of typhoid patients by means of morphological investigation techniques, immunofluorescent and electron microscopy. This suggests that typhoid granulomas form the basis of S. typhi primary carriership complicated by the relapses of this infection in cases of weakened cell-mediated immunity, which is proved by a decrease in the level of T-lymphocytes and by increased leukocyte migration index in relapses of typhoid fever and in S. typhi primary carriership. At the same time, the formation of S. typhi secondary carriership occurs in the process of the colonization of the altered organs and tissues of the body by S. typhi. This secondary carriership differs from the primary one by a number of pathogenetic signs. The detailed characterization of these two forms of S. typhi carriership is presented.

Opisthorchiatic solitary intrahepatic cyst.

Eleven patients were documented as having opisthorchiatic solitary intrahepatic cyst by roentgenography--P.T.C., identification of opisthorchis ova in the bile during P.T.C. and operation. This clinical entity should be differentiated from other forms of intrahepatic cyst. We believe that it is an acquired form. The formation of opisthorchiatic solitary intrahepatic cyst is related to lower biliary tract obstruction and high pressure in the biliary system. Fever, pain in the right upper quadrant and jaundice are the major manifestations of opisthorchiatic solitary intrahepatic cyst. Surgical intervention is the therapeutic method.