Metformin Reduces Blood Glucose in Treatment-Naive Type 2 Diabetes by Altering the Gut Microbiome.

BACKGROUND: Our aim in this study was to better understand the causality of metformin and gut microbiome in the treatment of type 2 diabetes (T2D). METHODS: This study was conducted on individuals with newly diagnosed and treatment-naive T2D. We used 16S rRNA sequencing to assess the effect of metformin on composition and diversity of the gut microbiota. We also compared the differences in relative abundance of gut microbiome at the genus level in patients with treatment-naive T2D before and after 2 months of metformin treatment. Spearman's rank correlation coefficient analysis was used to identify genus abundance in relation to blood glucose and related factors. RESULTS: Metformin significantly reduced blood glucose and levels of the related factors in treatment-naive individuals with T2D after 2 months of treatment. The 16S rRNA sequencing showed that metformin treatment altered composition and diversity of gut microbiome. Megamonas and Klebsiella in the T2D groups were significantly higher compared with the control group. Metformin treatment caused a significant reduction in Megamonas and Klebsiella. Spearman's rank correlation coefficient analysis showed a significant positive correlation between Megamonas and blood glucose, glycated hemoglobin (A1C), serum fructosamine and alanine aminotranferase (ALT). Klebsiella showed a significant positive correlation between A1C and ALT. CONCLUSION: Metformin reduces blood glucose in T2D by interacting with different gut bacteria, possibly Megamonas and Klebsiella pneumoniae.

Integrating nested PCR with high-throughput sequencing to characterize mutations of HBV genome in low viral load samples.

Due to the low viral load of hepatitis B virus (HBV) in plasma samples, conventional techniques have limitations to the detection of antiviral resistance mutations. To solve the problem, we developed a fast, highly sensitive, and accurate method to sequence the HBV whole-genome sequencing in plasma samples which had various viral loads from very low to high.Twenty-one plasma samples were collected from patients who were carriers of HBV from the Hangzhou First People's Hospital. Two pairs of conserved, overlapping, nested primers were used to amplify and sequence the whole HBV genome in 8 plasma samples with different viral loads. High-throughput sequencing was performed on Illumina MiSeq platform. Concomitantly, 3 samples were directly sequenced without PCR amplification. We compared amplicon-sequencing with direct sequencing to develop a method for amplifying and characterizing the whole genome of HBV.HBV genome was amplified from all samples and verified by Sanger sequencing, regardless of the viral loads. Sequencing results revealed that only a few reads were mapped to the HBV genome following direct sequencing, while the amplicon-sequencing reads had a good coverage and depth. We identified 50 intrahost single nucleotide variations (iSNVs), 14 of which were low frequency mutations. Interestingly, iSNVs were more common in low viral load samples than in high viral load samples, and mutations in the reverse transcriptase (RT) region were most prevalent.We conclude that amplicon-sequencing is not only a practical method to detect HBV infection with a high sensitivity and accuracy but also enables to detect mutations in the HBV genome in low viral load samples from HBV-infected patients. Thus, our findings provide a new diagnosis method of HBV infection, which is capable of detection of low frequent mutations in low viral load samples.

Molecular Epidemiology of Clostridium difficile Infection in Hospitalized Patients in Eastern China.

Few studies on risk factors for and transmission of Clostridium difficile infection (CDI) in China have been reported. A cross-sectional study was conducted for 3 years in eastern China. Consecutive stool specimens from hospitalized patients with diarrhea were cultured for C. difficile. C. difficile isolates from these patients then were analyzed for toxin genes, genotypes, and antimicrobial resistance. A severity score for the CDI in each patient was determined by a blinded review of the medical record, and these scores ranged from 1 to 6. A total of 397 out of 3,953 patients (10.0%) with diarrhea were found to have CDI. Severity of CDI was mild to moderate, and the average (± standard deviation) severity score was 2.61 ± 1.01. C. difficile was isolated from stool specimens in 432 (10.9%) of all the patients who had diarrhea. C. difficile genotypes were determined by multilocus sequence analysis and PCR ribotyping; sequence type 37 (ST37)/ribotype 017 (RT017) (n = 68, 16.5%) was the dominant genotype. Eleven patients (16.2%) with this genotype had a CDI severity score of 5. Overall, three RTs and four STs were predominant; these genotypes were associated with significantly different antimicrobial resistance patterns in comparison to all genotypes (χ2 = 79.56 to 97.76; P < 0.001). Independent risk factors associated with CDI included age greater than 55 years (odds ratio [95% confidence interval], 26.80 [18.76 to 38.29]), previous hospitalization (12.42 [8.85 to 17.43]), previous antimicrobial treatment within 8 weeks (150.56 [73.11 to 310.06]), hospital stay more than 3 days before sampling (2.34 [1.71 to 3.22]), undergoing chemotherapy (3.31 [2.22 to 4.92]), and undergoing abdominal surgery (4.82 [3.54 to 6.55]). CDI is clearly a problem in eastern China and has a prevalence of 10.0% in hospitalized patients. Among risk factors for CDI, the advanced age threshold was younger for Chinese patients than that reported for patients in developed countries.

Preliminary exploration of HLA-A 1101-restricted human cytomegalovirus glycoprotein B-specific CD8⁺ T cells in allogeneic stem-cell transplant recipients.

T-cell responses directed against human cytomegalovirus (HCMV) glycoprotein B (gB) contribute to protective immunity against HCMV infection in both animal models and humans. However, the gB-specific human CD8(+) T cell responses remain poorly understood. gB antigen-specific CD8(+) T cells were stained with seven major histocompatibility complex (MHC)-peptide pentamers in 16 human leukocyte antigen (HLA)-A 1101-positive, HCMV-seropositive patients following hematopoietic stem cell transplantation (HSCT). Of these seven pentamers, the most frequent CD8(+) T-cell responses were directed against the gB332-340 peptide. These gB332-340-specific CD8(+) T cells were strongly associated with the presence of plasma HCMV immunoglobulin M in all HSCT recipients and exhibited a probable causal relationship with the level of pp65 antigenemia. Together, these data suggest a role for gB332-340-specific CD8(+) T cells in HCMV reactivation after HSCT. Furthermore, the pentamer assay may be valuable in detecting antigen-specific CD8(+) T cells.

Evaluation of human cytomegalovirus-specific CD8+ T-cells in allogeneic haematopoietic stem cell transplant recipients using pentamer and interferon-γ-enzyme-linked immunospot assays.

BACKGROUND: Reactivation of latent human cytomegalovirus (HCMV) is a frequent complication following allogeneic haematopoietic stem cell transplantation (HSCT). Evaluation of the quantity and function of HCMV-specific CD8+ T-cell responses after HSCT may play a crucial role in the prevention of HCMV reactivation. OBJECTIVES: To investigate the mechanism of HCMV-specific T-cell immune responses after HSCT in HCMV-specific CD8+ T cells. STUDY DESIGN: HCMV-specific CD8+ T cells were quantified using human leucocyte antigen (HLA) pentamer staining and functionally analysed by interferon-γ-enzyme-linked immunospot (IFN-γ-ELISPOT) assay with a pp65495-503 peptide in recipients four years after HSCT. RESULTS: The absolute number of pp65495-503-specific CD8+ T cells did not differ significantly (p>0.05) between samples with antigenaemia and those without antigenaemia given a mean of 54.5/µl and 40.5/µl, respectively, in 21 HLA-A* 0201 patients after HSCT. The level of pp65495-503-specific CD8+ T cells>20/µl of peripheral blood was maintained 90 days after transplantation. There was a significant difference in the spot count of IFN-γ-secreting T cells between samples with antigenaemia (mean, 507/2.5×10(5)PBMCs) and those without antigenaemia (mean, 216/2.5×10(5)PBMCs; p<0.05). CONCLUSION: pp65495-503-specific CD8+ T cells may not be sufficient to control HCMV reactivation in recipients after HSCT. However, the combination of pentamer and IFN-γ-ELISPOT assays may be valuable for evaluating HCMV-specific CD8+ T cells. Further studies on HCMV-specific T-cell immune responses continue to be performed for the prevention of persistent HCMV reactivation.