Metagenomic Analysis of Biliary Microbial Flora in Patients with Gallbladder Cancer or Gallstones-Associated Chronic Cholecystitis.

Biliary dysbiosis is associated with gallbladder cancer (GBC). We aimed to look for biliary bacteria specifically detected in GBC patients. We used 16S rRNA-based metagenomic analysis to elucidate biliary microbiota in 30 GBC and 30 gallstones-associated chronic cholecystitis patients. Relative abundance of five genera, Streptococcus, Enterococcus, Halomonas, Escherichia and Caulobacter was significantly associated with GBC. Of 15-species, 7 were detected significantly higher in GBC, Streptococcus anginosus, Streptococcus constellatus, Streptococcus intermedius, Actinomyces bowdenii, Actinomyces israelii, Actinomyces gerencseriae, and Escherichia fergusonii were biosafety level-2 infectious bacteria; other 8 species were biosafety level-1 bacteria. These bacterial species may be involved in pathogenesis of GBC.

Roles of Salmonella typhi and Salmonella paratyphi in Gallbladder Cancer Development.

BACKGROUND: Typhoid (Salmonella typhi and paratyphi) carriers and gall bladder cancer (GBC) are endemic in northern India. Results of previous studies about association of typhoid carriers with GBC are inconsistent. We studied antibodies against Salmonella typhi and paratyphi in serum samples of patients with GBC. METHODS: We performed modified Widal test for antibodies against Salmonella typhi (Vi and O) and Salmonella paratyphi (AO and BO) antigens in patients with GBC (n=100), xanthogranulomatous cholecystitis (XGC, n=24), chronic cholecystitis (CC, n=200) and healthy controls (HC, n=200). RESULTS: Serum antibodies against Salmonella were more frequently positive in GBC (22%) and XGC (29%), particularly in males in age ≥50 years (GBC: 47% and XGC: 50%) vs. HC (0) (p <0.01). Vi antibody was more common in GBC (13%, OR:9.8) and XGC (8%, OR:5.9) than HC (2%). O antibody was more common in GBC (8%, OR: 8.6) and XGC (8%, OR: 9.0) than HC (1%). O antibody was also more common in males with GBC (12%) than CC (1%) and HC (1%) (P=0.02 and p <0.001, respectively). AO (6%) and BO (4%) antibodies were detected in GBC, particularly in males, than HC (0), (p <0.01). Salmonella antibodies were more frequent in GBC with GS than those without GS (50% vs. 20%, OR=3.94, P=0.01). CONCLUSIONS: Salmonella carrier state was more common in GBC and XGC, particularly in elderly males than HC. The Vi antibody was more common in GBC and XGC than HC. Salmonella infection was more common in GBC with GS than those without GS.

Risk factors for gallbladder cancer development in northern India: A gallstones-matched, case-control study.

Background & objectives: A high incidence of gallbladder cancer (GBC) is observed in northern India. This study was aimed to identify the factors involved in developing GBC in this region. Methods: A gallstones-matched, case-control study was conducted in northern India. Ninety nine patients with GBC and gallstones (33 men and 66 women, mean age of 51.4 yr) comprised the case group, while 99 patients with cholelithiasis (40 men and 59 women, mean age of 45.7 yr) comprised the control group. All participants were interviewed to complete 183 questionnaire items that included 105 food items. Potential risk factors were identified using a multivariate analysis adjusted for age and sex. Significant risk factors were identified using a stepwise logistic-regression analysis. Results: Age (≥50 yr), education (illiterate), socioeconomic status (≤below poverty line), bowel habits (≤once a day), hypertension history, hypotensive drug use, non-vegetarian diet, use of firewood for cooking, tap water drinking, hand pump water drinking and high consumption of coffee and sweets were identified as the potential risk factors. In women, factors included menarche (<13 yr), number of pregnancies (≥3 pregnancies) and parity (≥3 babies). Of these factors, age, education, bowel habits, tap water drinking and multiple pregnancy and/or multiparity were identified as significant risk factors, whereas a high consumption of coffee and sweets or hypotensive drug use and/or hypertension history were protective factors. Interpretation & conclusions: Poor bowel habits and drinking unsafe water appear to be the main risk factors for developing GBC. These are, however, modifiable factors which are capable of decreasing the risk of GBC in the north Indian population.

Carcinogen Metabolism Pathway and Tumor Suppressor Gene Polymorphisms and Gallbladder Cancer Risk in North Indians: A Hospital-Based Case-Control Study.

BACKGROUND: Carcinogen metabolism pathway and tumor suppressor gene polymorphisms have been reported to be associated with increased gallbladder cancer risk. However, the association of genetic variants and gallbladder cancer risk in Indians are not well studied. We examined whether genetic polymorphisms of metabolic enzymes cytochrome P450 1A1 and glutathione S-transferase and tumor suppressor gene p53 (TP53) are associated with an increased risk of gallbladder cancer in North Indians. METHODS: This hospital-based case-control study was conducted in 96 gallbladder cancer patients with gallstones (cases) and 93 cholelithiasis patients (controls) at the Sanjay Gandhi Postgraduate Institute of Medical Sciences in Lucknow, India from July 2014 through May 2017. Genomic DNA was extracted from white blood cells of each patient using a simple salting-out procedure. The genotypic frequencies of CYP1A1 rs4646903, CYP1A1 rs1048943, and TP53 rs1042522 polymorphisms were investigated using TaqMan SNP Genotyping Assay and GSTM1 and GSTT1 polymorphisms were analyzed using the multiplex PCR assay. RESULTS: The frequency of CC genotype of TP53 rs1042522 polymorphism was 27.1% (26/96) in cases and 12.9% (12/93) in controls. The CC genotype was associated with an increased risk of gallbladder cancer in North Indians (age- and sex-adjusted odds ratio, 2.81; 95% confidence interval, 1.19-6.61; P = 0.02). No significant differences in genotypic and allelic frequencies of the metabolic pathway gene polymorphisms were found between cases and controls. CONCLUSIONS: Our data provide preliminary evidence that the CC genotype of the TP53 rs1042522 polymorphism may be associated with an increased risk of gallbladder cancer in North Indians.

Tumor Protein p53 and K-ras Gene Mutations in Peruvian Patients with Gallbladder Cancer

Background: Recent studies have shown that genetic alterations are associated with the effect of patient geographic location on gallbladder cancer development. Peru has a high incidence of gallbladder cancer, but causative factors have not yet been identified. We examined the frequency of mutations in TP53 and K-ras genes in Peruvian patients with gallbladder cancer, and compared this with data from Bolivia, Hungary, Chile, and Japan, which have a high gallbladder cancer incidence. Methods: DNA was extracted from formalin-fixed paraffin-embedded gallbladder tissue sections of 30 gallbladder cancer patients (9 men and 21 women) obtained using microdissection. Mutations in exons 5 to 8 of TP53 and codons 12, 13, and 61 of K-ras were examined using direct sequencing. Results: TP53 mutations were observed in 10 (33.3%) of patients, but K-ras mutations were absent. Nine (90%) TP53 mutations were point mutations (7 missense and 2 silent mutations), and the most frequent substitution was a G:C to A:T transition. G:C to A:T transitions at the CpG site or G:C to T:A transversions were found in one patient each. No significant differences were found in the frequency of TP53 and K-ras mutations among patients in the 5 countries. Conclusions: Our findings suggest that endogenous mechanisms and exogenous carcinogens may affect the carcinogenic process in Peruvian gallbladder cancer patients, similar to that in Bolivian patients. Further studies with a larger sample size are needed to clarify these findings.

Plasma Helicobacter pylori Antibody Titers and Helicobacter pylori Infection Positivity Rates in Patients with Gallbladder Cancer or Cholelithiasis: a Hospital-Based Case-Control Study

Objective: Gallbladder cancer is the commonest gastrointestinal cancer in northern Indian women. Some studies have examined the association between Helicobacter pylori infection and gallbladder cancer risk, but findings have been inconsistent. We aimed to examine the association between H. pylori infection and gallbladder cancer in Indian people. Materials and Methods: We conducted a hospital-based case-control study including 100 gallbladder cancer patients with gallstones who were 32 to 79 years old (cases; 72 women and 28 men), and 100 cholelithiasis patients aged 14 to 75 years (controls; 65 women and 35 men). All patients had a diagnosis of gallbladder cancer or cholelithiasis at the Sanjay Gandhi Post Graduate Institute of Medical Sciences in Lucknow having a high gallbladder cancer incidence in northern India, from May 2014 through July 2017. Plasma samples were collected from all patients before surgical treatment. Plasma H. pylori antibody titer was measured by the latex agglutination method and an autoanalyzer. H. pylori infection was defined as antibody titer ≥10 U/mL. Plasma antibody titers and H. pylori infection positivity rates were compared between cases and controls. Results: Mean plasma antibody titers (standard deviation, range) were 11.1 U/mL (11.6, 0­78) in cases and 13.6 U/mL (23.0, 1­164) in controls. H. pylori infection positivity rates were 41% and 42% in cases and controls, respectively. No significant differences in antibody titers or H. pylori infection positivity rates were found between cases and controls. Conclusions: We found no evidence of H. pylori infection as an important risk factor for gallbladder cancer in Indian people.

Metagenomics of Microbial Communities in Gallbladder Bile from Patients with Gallbladder Cancer or Cholelithiasis

Salmonella typhi and Helicobacter infections have been shown to increase risk of gallbladder cancer (GBC), but findings have been inconsistent. Other bacterial infections may also be associated with GBC. However, information on microbial pathogens in gallbladder bile of GBC patients is scarce. We aimed to investigate the microbial communities in gallbladder bile of patients with GBC and cholelithiasis (CL). Seven GBC patients and 30 CL patients were enrolled in this study. Genomic DNA was extracted from bile and the V3-V4 region of 16S rRNA was amplified. The sequencing results were compared with the 16S database, and the bacteria were identified by homology searches and phylogenetic analysis. DNA was detected in the bile of three GBC (42.9%; Bolivia, 1; Chile, 2) and four CL patients (13.3%; Bolivia, 1; Chile, 3). Of the 37 patients, 30 (81.1%) were negative and unable to analyze. Salmonella typhi and Helicobacter sp. were not detected in bile from any GBC patients. As the predominant species, Fusobacterium nucleatum, Escherichia coli, and Enetrobacter sp. were detected in bile from GBC patients. Those in bile from CL patients were Escherichia coli, Salmonella sp., and Enerococcus gallinarum. Escherichia coli was detected in bile samples from both GBC and CL patients. Whether the bacteria detected in bile from GBC patients would associated with the development of GBC warrant further investigation.

Effect of Mass Stool Examination and Mass Treatment For Decreasing Intestinal Helminth and Protozoan Infection Rates in Bolivian Children: A Cross-Sectional Study.

Bolivia is one of the countries with a high intestinal helminth and protozoan infection rate. Despite the high prevalence of the parasitic infection, nationwide preventive measures for Bolivian children have not yet been implemented. We evaluated the effect of mass stool examination and treatment as a strategy for decreasing the infection rate. This study was conducted between 2013 and 2015 in children aged 2-18 years. A total of 2,033 stool samples (575 in 2013, 815 in 2014 and 642 in 2015) were collected and examined using the formalin-ether medical sedimentation method. As an anthelminthic medicine, nitazoxanide was given to all infected children within 2 months post-examination, each year. The effect of mass stool examination and treatment was evaluated based on the changes in the overall or individual parasitic infection rates during the study period. The overall parasitic infection rate decreased significantly from 65.2% in 2013 to 43.0% in 2015; a 22.2 percentage point decrease (P<0.001). Protozoan infection accounted for a large portion of the parasitic infections, in the following rates: 62.4% in 2013, 49.3% in 2014, and 41.0% in 2015. The rate of the most common helminth infection, Hymenolepis nana, decreased significantly from 9.0% in 2013 to 6.4% in 2014 to 3.4% in 2015 (P<0.001). Prevalence of the most common pathogenic protozoan infection, Entamoeba histolytica, decreased significantly from 19.0% in 2013 to 3.0% in 2015 (P<0.001). Conversely, the rate of Giardia intestinalis increased significantly from 16.5% in 2013 to 21.2% in 2015 (P<0.01). Mass stool examination and treatment for intestinal helminth and protozoan infections was effective for decreasing the overall parasitic infection rate in the study population, excluding Giardia intestinalis. Further studies on the long-term effect of mass stool examination and treatment for decreasing all intestinal parasitic infection rates in Bolivian children are needed.

Lack of an Apparent Association between Mycotoxin Concentrations in Red Chili Peppers and Incidence of Gallbladder Cancer in India : an Ecological Study.

Our recent studies conducted in South America have shown that mycotoxin contamination of red chili peppers (RCPs) may be associated with an increased risk of gallbladder cancer (GBC). Whether this relationship exists in India, a country with a high incidence of GBC and high consumption of RCPs, is unclear. We therefore measured concentrations of aflatoxins (AFs) and ochratoxin A (OTA) in RCPs from areas of low, medium, and high incidence of GBC in India, and compared these concentrations with GBC incidence in each area. Twentyone RCP samples were collected from nine cities (eight from a lowincidence area, five from a mediumincidence area, and eight from a highincidence area). Concentrations of AFs and OTA were measured using highperformance liquid chromatography. No significant differences in mean concentrations of AFs and OTA were found in the three areas. AFB1 levels in the lowincidence area (10.81 ?g/kg) and highincidence area (12.00 ?g/kg) were more than 2.2 and 2.4 times higher compared with the maximum permitted level of AFB1 in spices (5.0 ?g/kg) set by the Commission of the European Communities, or that (4.4 ?g/kg) obtained in our previous study in Chile. Our results show that the mean concentrations of mycotoxins in RCPs are similar among the three areas in India with different incidences of GBC. Further studies with human subjects are needed to evaluate any association between AFB1 and GBC.

CYP1A1, GSTM1, GSTT1 and TP53 Polymorphisms and Risk of Gallbladder Cancer in Bolivians.

The Plurinational State of Bolivia (Bolivia) has a high incidence rate of gallbladder cancer (GBC). However, the genetic and environmental risk factors for GBC development are not well understood. We aimed to assess whether or not cytochrome P450 (CYP1A1), glutathione S-transferase mu 1 (GSTM1), theta 1 (GSTT1) and tumor suppressor protein p53 (TP53) genetic polymorphisms modulate GBC susceptibility in Bolivians. This case-control study covered 32 patients with GBC and 86 healthy subjects. GBC was diagnosed on the basis of histological analysis of tissues at the Instituto de Gastroenterologia Boliviano-Japones (IGBJ); the healthy subjects were members of the staff at the IGBJ. Distributions of the CYP1A1 rs1048943 and TP53 rs1042522 polymorphisms were assayed using PCR-restriction fragment length polymorphism assay. GSTM1 and GSTT1 deletion polymorphisms were detected by a multiplex PCR assay. The frequency of the GSTM1 null genotype was significantly higher in GBC patients than in the healthy subjects (odds ratio [OR], 2.35; 95% confidence interval [CI], 1.03-5.37; age-adjusted OR, 3.53; 95% CI, 1.29-9.66; age- and sex-adjusted OR, 3.40; 95% CI, 1.24-9.34). No significant differences were observed in the frequencies of CYP1A1, GSTT1, or TP53 polymorphisms between the two groups. The GSTM1 null genotype was associated with increased GBC risk in Bolivians. Additional studies with larger control and case populations are warranted to confirm the association between the GSTM1 deletion polymorphism and GBC risk suggested in the present study.

Ochratoxin A Contamination of Red Chili Peppers from Chile, Bolivia and Peru, Countries with a High Incidence of Gallbladder Cancer.

Our previous study detected aflatoxins in red chili peppers from Chile, Bolivia, and Peru, each of which have a high incidence of gallbladder cancer (GBC). Since the aflatoxin B1 concentration was not so high in these peppers, it is important to clarify the presence of other mycotoxins. Here we attempted to determine any associations between the concentrations of aflatoxins and ochratoxin A (OTA) in red chili peppers, and the corresponding GBC incidences. We collected red chili peppers from three areas in Peru: Trujillo (a high GBC incidence area), Cusco (an intermediate GBC incidence area), and Lima (a low GBC incidence rate), and from Chile and Bolivia. Aflatoxins and OTA were extracted with organic solvents. The concentrations of aflatoxins B1, B2, G1, and G2, and OTA were measured by high-performance liquid chromatography. The values obtained were compared with the incidence of GBC in each area or country. All of the red chili peppers from the three areas showed contamination with aflatoxins below the Commission of the European Communities (EC) recommended limits (5 µg/kg), but the OTA contamination of two samples was above the EC recommended limit (15 µg/kg). The mean concentrations of OTA in the peppers from Chile (mean 355 µg/kg, range <5-1,059 µg/kg) and Bolivia (mean 207 µg/kg, range 0.8-628 µg/kg), which has a high incidence of GBC, were higher than that in Peru (14 µg/kg, range <5-47 µg/kg), which has an intermediate GBC incidence. The OTA contamination in the red chili peppers from Chile, Bolivia, and Peru was stronger than that of aflatoxins. Our data suggest that OTA in red chili peppers may be associated with the development of GBC.

Aflatoxin contamination of red chili pepper from Bolivia and Peru, countries with high gallbladder cancer incidence rates.

Chilean red chili peppers contaminated with aflatoxins were reported in a previous study. If the development of gallbladder cancer (GBC) in Chile is associated with a high level of consumption of aflatoxin-contaminated red chili peppers, such peppers from other countries having a high GBC incidence rate may also be contaminated with aflatoxins. We aimed to determine whether this might be the case for red chili peppers from Bolivia and Peru. A total of 7 samples (3 from Bolivia, 4 from Peru) and 3 controls (2 from China, 1 from Japan) were evaluated. Aflatoxins were extracted with acetonitrile:water (9:1, v/v) and eluted through an immuno-affinity column. The concentrations of aflatoxins B1, B2, G1, and G2 were measured using high-performance liquid chromatography (HPLC), and then the detected aflatoxins were identified using HPLC-mass spectrometry. In some but not all of the samples from Bolivia and Peru, aflatoxin B1 or aflatoxins B1 and B2 were detected. In particular, aflatoxin B1 or total aflatoxin concentrations in a Bolivian samples were above the maximum levels for aflatoxins in spices proposed by the European Commission. Red chili peppers from Bolivia and Peru consumed by populations having high GBC incidence rates would appear to be contaminated with aflatoxins. These data suggest the possibility that a high level of consumption of aflatoxin-contaminated red chili peppers is related to the development of GBC, and the association between the two should be confirmed by a case-control study.