Gut microbiome is associated with the clinical response to anti-PD-1 based immunotherapy in hepatobiliary cancers

BackgroundThe gut microbiome is associated with the response to immunotherapy for different cancers. However, the impact of the gut microbiome on hepatobiliary cancers receiving immunotherapy remains unknown. This study aims to investigate the relationship between the gut microbiome and the clinical response to anti-programmed cell death protein 1 (PD-1) immunotherapy in patients with advanced hepatobiliary cancers.MethodsPatients with unresectable hepatocellular carcinoma or advanced biliary tract cancers who have progressed from first-line chemotherapy (gemcitabine plus cisplatin) were enrolled. Fresh stool samples were collected before and during anti-PD-1 treatment and analyzed with metagenomic sequencing. Significantly differentially enriched taxa and prognosis associated taxa were identified. The Kyoto Encyclopedia of Genes and Genomes database and MetaCyc database were further applied to annotate the differentially enriched taxa to explore the potential mechanism of the gut microbiome influencing cancer immunotherapy.ResultsIn total, 65 patients with advanced hepatobiliary cancers receiving anti-PD-1 treatment were included in this study. Seventy-four taxa were significantly enriched in the clinical benefit response (CBR) group and 40 taxa were significantly enriched in the non-clinical benefit (NCB) group. Among these taxa, patients with higher abundance of Lachnospiraceae bacterium-GAM79 and Alistipes sp Marseille-P5997, which were significantly enriched in the CBR group, achieved longer progression-free survival (PFS) and overall survival (OS) than patients with lower abundance. Higher abundance of Ruminococcus calidus and Erysipelotichaceae bacterium-GAM147 enriched in the CBR group was also observed in patients with better PFS. In contrast, worse PFS and OS were found in patients with higher abundance of Veillonellaceae, which was significantly enriched in the NCB group. Functional annotation indicated that the taxa enriched in the CBR group were associated with energy metabolism while the taxa enriched in the NCB group were associated with amino acid metabolism, which may modulate the clinical response to immunotherapy in hepatobiliary cancers. In addition, immunotherapy-related adverse events were affected by the gut microbiome diversity and relative abundance.ConclusionsWe demonstrate that the gut microbiome is associated with the clinical response to anti-PD-1 immunotherapy in patients with hepatobiliary cancers. Taxonomic signatures enriched in responders are effective biomarkers to predict the clinical response and survival benefit of immunotherapy, which might provide a new therapeutic target to modulate the response to cancer immunotherapy.

Comparison of Molecular and Parasitological Methods for Diagnosis of Human Trichostrongylosis

Human trichostrongyliasis is a zoonotic disease that is prevalent among rural populations in some countries. This study was performed to evaluate various parasitological methods and polymerase chain reaction (PCR) for the diagnosis of human trichostrongyliasis. A total of 206 fresh stool samples were collected from residents of endemic villages of Northern Iran. All samples were examined using conventional parasitological methods, including wet mount, formalin ethyl acetate concentration (FEAC), agar plate culture (APC), Harada–Mori culture (HMC), and Willis, along with the PCR technique. Among the total of 206 individuals examined, 72 people (35%) were found infected with Trichostrongylus species using combined parasitological methods. By considering the combined results of parasitological methods as the diagnostic gold standard, the Willis technique had a sensitivity of 91.7% compared with 52.8% for the APC, 40.3% for the HMC, 37.5% for FEAC, and 5.6% for the wet mount technique. The diagnostic specificity of all the parasitological methods was 100%. Furthermore, the PCR method detected Trichostrongylus spp. DNA in 79 fecal samples (38.3%) with a sensitivity of 97.2% and a specificity of 93.3%. According to the current findings, the Willis method was more sensitive than are the other parasitological methods in the diagnosis of human trichostrongyliasis. However, the PCR assay was more sensitive and more reliable in the detection of human trichostrongyliasis in comparison with the parasitological methods.

Comparative genomic characterization of multidrug-resistant Citrobacter spp. strains in Fennec fox imported to China

Background To investigate the antimicrobial profiles and genomic characteristics of MDR-Citrobacter spp. strains isolated from Fennec fox imported from Sudan to China. Methods Four Citrobacter spp. strains were isolated from stool samples. Individual fresh stool samples were collected and subsequently diluted in phosphate buffered saline as described previously. The diluted fecal samples were plated on MacConkey agar supplemented with 1 mg/l cefotaxime and incubated for 20 h at 37 °C. Matrix-assisted laser desorption/ionization–time of flight mass spectrometry (MALDI–TOF–MS) was used for identification. Antimicrobial susceptibility testing was performed using the broth microdilution method. Whole-genome sequencing was performed on an Illumina Novaseq-6000 platform. Acquired antimicrobial resistance genes and plasmid replicons were detected using ResFinder 4.1 and PlasmidFinder 1.3, respectively. Comparative genomic analysis of 277 Citrobacter genomes was also performed. Results Isolate FF141 was identified as Citrobacter cronae while isolate FF371, isolate FF414, and isolate FF423 were identified as Citrobacter braakii. Of these, three C. braakii isolates were further confirmed to be extended-spectrum β-lactamases (ESBL)-producer. All isolates are all multidrug resistance (MDR) with resistance to multiple antimicrobials. Plasmid of pKPC-CAV1321 belong to incompatibility (Inc) group. Comparative genomics analysis of Citrobacter isolates generated a large core-genome. Genetic diversity was observed in our bacterial collection, which clustered into five main clades. Human, environmental and animal Citrobacter isolates were distributed into five clusters. Conclusions To our knowledge, this is the first investigation of MDR-Citrobacter from Fennec Fox. Our phenotypic and genomic data further underscore the threat of increased ESBL prevalence in wildlife and emphasize that increased effort should be committed to monitoring the potentially rapid dissemination of ESBL-producers with one health perspective.

Fecal screening of Dientamoeba fragilis: relative efficacy of permanent smear and culture

Dientamoeba fragilis (D. fragilis) is an enteric trichmonad protozoan parasite that remains obscure and neglected. The aim of this study is to detect D. fragilis as a neglected pathogen in children aged 6-12 years old complaining of gastrointestinal illness by stool culture and light microscopy with comparison between the results of both techniques. A total of 100 fresh stool samples were included in this current study. All specimens were subjected to microscopic examination using iron- hematoxylin stained stool smears and stool culture using a Loeffler’s culture medium. Culture detected 2 positive stool samples (2%) while microscopy detected (1%). Sensitivities of culture and microscopy were 100% and 50% respectively. Specificity of culture and microscopy were 100% and 95% respectively. There is a moderate agreement between culture and microscopy (K = 0.4). In conclusion, culture had a high performance compared to microscopy in the diagnosis of D. fragilis infection. Culture can be applied for routine diagnosis for the detection of D. fragilis in clinical samples.

Pilot Sub-Study of the Effect of Hepatitis C Cure by Glecaprevir/Pibrentasvir on the Gut Microbiome of Patients with Chronic Hepatitis C Genotypes 1 to 6 in the Mythen Study

In this small pilot sub-study, longitudinal gut microbiota composition changes, after successful treatment of hepatitis C virus (HCV) with the co-formulated glecaprevir/pibrentasvir (GLE/PIB), were analyzed before treatment (baseline) and 12 weeks post-treatment. Participating patients provided a fresh stool sample the week before their study visit, from which microbial DNA was extracted and sequenced for the 16S rRNA region in an Illumina MiSeq2 platform. Microbial and statistical analyses were conducted to determine the alpha-diversity (number of different taxa within a sample) and beta-diversity (number of overlapping taxa between samples). Stool samples from 58 patients were eligible for analysis. There were 27 patients with HCV genotype 1, 10 with genotype 2, 16 with genotype 3, and 5 with genotype 4. No statistically significant differences in gut microbiota diversity, species richness, or microbial community pattern were found at baseline and at post-treatment Week 12. Lack of statistically significant differences remained consistent in further analysis by demographic and baseline disease characteristics. Surprisingly, no statistically significant changes in alpha- and beta-diversity were seen in the microbiota after GLE/PIB treatment, though there was a trend toward less richness over time. Further investigation is needed into this unexpected outcome to better understand the role of HCV treatment and the gut microbiota.

Detection of Closteridium difficile in patients with Hematological Malignancies: A Stool Based Immunochromatographic study

Background: Diarrhea is a common problem in patients with hematologic disease and in patients with immuno compromising conditions. Objective: The aim of the study was to evaluate the presence risk factors that leading to acquire infection with Cl. difficile in children infected sever diarrhea in and immuno compromised patients. Material and Methods: The study was performed on freshly collected stool samples among 32 patients (13 females,19 males) with acute diarrheal in children their ages (1.5 months – 1.3 years) admitted to Pediatric Teaching Hospitals and adults admitted to Baghdad Teaching Hospital (patients with low cellular immunity like ALL, AML (20-75years) from April 2017 to April 2019. A questionnaire was completed for each patient name, age, gender, clinical symptoms like fever, diarrhea, constipation and, abdominal pain. The criteria included also, the risk factors that leading to suppression of cellular immunity. Fresh stool samples were tested by immunochromatographic assay for antigenic detection of Clostridium difficile Ag. Results: C. Idifficile antigen was identified only in three stool samples of male their mean ± S.D. was (24.75-24.37) from 32 patients (13 females,19 males) their ages rang (1.5months-75years), seven of them suffered blood malignancies (such as AML, ALL), while 21patients (seven of them babies) suffered from acute diarrhea with different causes (E. histolytica and fungal infections). All patients had fever, flatulence and abdominal patients, while 4 of patients with low cellular immunity. Conclusion: Cl. diffcile antigen present only few number of patients in three stool samples of maleswho suffered from sever diarrhea, or ALL (acute lymphocytic leukemia), or NHL (non-hodgkinse lymphoma).

Prevalence of intestinal parasites among children attending outpatient department of Kanti Children’s Hospital, Kathmandu, Nepal

Intestinal parasitic infection is one of the main causes of morbidity and mortality in developing countries, especially among children. Even minimum infection of parasites in children may have negative effects on growth, iron deficiency anemia, perceiving function, and impaired cognition. The main objective of this study was to determine the prevalence of intestinal parasites and associated factors among the children attending the Outpatient Department of Kanti Children’s Hospital, Kathmandu for various illnesses. The research was carried out from March to May 2018. A total of 300 fresh stool samples were collected in clean, dry and screw-capped plastic vials and were studied for the presence of intestinal parasites using the direct smear and concentration methods. Children or their parents were interviewed using standard questionnaires. The overall prevalence of intestinal parasites was 25.67%. The protozoan infestation was found in 22.67% of cases, while helminthic infestation was found only in 3% of cases. No double infestation was detected. The predominant parasite was Entamoeba histolytica (14%) followed by Giardia lamblia (8. 67%). The prevalence among female (32.11%) was greater than male (21.99%). The infection was found higher in low age of children, using underground water as a source of drinking water and hardly cut their nail in a regular fashion, whereas those children followed regular hand washing habit, defecation in toilet, parent’s occupation, use of antihelminthic drugs and treatment method had low infection of intestinal parasitic infection. All these evidences have shown that there should be an effective implementation of intervention activities to control and cure the spread of parasites associated infections among children.

Aboriginal Bacterial Flora in the Uricase-Deficient Rat Gut is Not the Main Factor Affecting Serum Uric Acid

The relationship between intestinal bacteria and hyperuricemia is a hot research topic. To better understand this relationship, uricase-deficient Sprague–Dawley rats (Kunming-DY rats) were used. The wild-type rats and Kunming-DY rats were used as controls. Kunming-DY rats were treated with ampicillin (90 mg/kg) and ciprofloxacin (150 mg/kg) for 5 days. Bacterial 16S rDNA in the fresh stool was sequenced, and the abundance was calculated. The rats’ serum uric acid (SUA) level was assayed, and the rats’ intake and output in 24 h were recorded. The bacterial diversity in three groups’ fresh stool was analyzed. The gut bacterial diversity and abundance changed in the Kunming-DY rats. More than 99% of bacteria were inhibited or killed by the combination of antibiotics. In contrast to each of the antibiotics alone, the combination of antibiotics lowered the Kunming-DY rats’ SUA level; it also caused mild diarrhea, which increased uric acid excretion through stool. These results suggested that the aboriginal gut bacteria in uricase-deficient rats play a minor role in determining the SUA levels. It is too early to conclude that aboriginal gut bacteria are a tempting target for lowering SUA levels.

Possible Association and Risk Factors of Blastocystis Infection and Colorectal Cancers in Western Iran

Background: Recently, infection has been considered one of the most important causes of cancers because a large number of cases of cancer with infectious origin was reported. Objectives: The present investigation aimed to evaluate the prevalence and risk factors of Blastocystis hominis infection in patients with colorectal cancer in comparison to healthy individuals. Methods: The present descriptive case-control study was performed on 67 healthy individuals and 67 patients with colorectal cancers attending the general hospitals of Lorestan Province, Western Iran from October 2017 to August 2018. Colorectal cancers were diagnosed by an experienced gastroenterologist. A fresh stool specimen was collected from each subject in a sterile labeled container. The collected specimens were tested microscopically using saline and iodine wet preparations, then stained with trichrome stain according to the manufacturer’s instruction to find the cases of B. hominis forms. The DNA of the samples was extracted and specific primer-polymerase chain reaction (PCR) was performed. Results: Among the colorectal cancer patients, B. hominis was found in 16 (23.9%) patients, whereas of 67 healthy participants, 6 (9%) cases were found, indicating a significant difference (P < 0.001) in the prevalence B. hominis among the participants in the case and control groups. By the multifactorial logistic regression models, agriculture activity (0.24; 95% CI: 0.075 - 0.809), as well as consumption of unwashed fruit and vegetables (0.136; 95% CI: 0.040 - 0.459), were significantly related to the prevalence of B. homonis infection. All 22 positive samples (16 patients and 6 (9%) healthy people) were also positive by PCR method, indicating the presence of B. hominis and accuracy of microscopic examination, extraction, and PCR reaction. Conclusions: The obtained findings revealed that B. hominis may strongly link with human colorectal cancers given novel information about the important role of B. hominis in the progress of colorectal cancer. Nevertheless, more investigations are required to obtain accurate information about this suggestion.

Carriage of extended-spectrum beta-lactamase-producing Enterobacteriaceae by healthy school children from two remote villages in western Cameroon

Carriage of extended-spectrum beta-lactamases (ESBL)-producing Enterobacteriaceae by healthy children can increase the risk of developing a lethal pathological infection. The objective of this study was to determine the rate of ESBL-producing Enterobacteriaceae carriage among children in remote villages in western Cameroon. We collected fresh stool samples from 110 healthy primary school children between 2 to 5 years old in two remote villages. The bacteria isolates were characterized using the Api 20E gallery, disc diffusion, and double-disc synergy test. Logistic regression analysis was used to determine the risk factors associated with the carriage of ESBL-producing Enterobacteriaceae. Data analysis indicated that a total of 24 children in 110 (22%) investigated were positive to ESBL-producing Enterobacteriaceae. Moreover, 24 (67%) out of 36 bacteria isolates were ESBL producers and 15 (61%) out of 24 being Escherichia coli. Other ESBL-producing bacteria were Klebsiella pneumoniae (3%) and Kluyvera spp (3%). We also isolated a small proportion of bacteria showing resistance to high-level cephalosporins, which overall represented 33% of the total bacteria isolates. Furthermore, risk factors associated with the carriage of ESBL-producing Enterobacteriaceae were the use of pesticides in agriculture and farming practice. The current result suggests that frequent contact to antibiotics is not the only reason for the development of resistance and confirm that resistance can be induced by chemicals from pesticide origin.