Partners of patients with ulcerative colitis exhibit a biologically relevant dysbiosis in fecal microbial metacommunities.

AIM: To investigate alterations in the fecal microbiome using 16S rRNA amplicon sequencing in couples in the same cohabitation environment. METHODS: Fecal samples were collected from eight ulcerative colitis (UC) patients and their healthy partners at Lishui People's Hospital, Zhejiang Province, China. DNA was extracted and the variable regions V3 and V4 of the 16S rRNA genes were PCR amplified using a two-step protocol. Clear reads were clustered into operational taxonomic units (OTUs) at the 97% sequence similarity level using UCLUST v1.2.22. The Wilcoxon rank-sum test (R v3.1.2) was used to compare inter-individual differences. Differences with a P value < 0.05 were considered statistically significant. RESULTS: Fecal microbial communities were more similar among UC patients than their healthy partners (P = 0.024). UC individuals had a lower relative abundance of bacteria belonging to the Firmicutes, especially Blautia, Clostridium, Coprococcus and Roseburia (P < 0.05). Microbiota dysbiosis was detected in UC patients and their healthy partners. Relevant genera included Akkermansiam, Bacteroides, Escherichia, Lactobacillales, Klebsiella and Parabacteroides. The enriched pathways in fecal samples of UC patients were related to lipid and nucleotide metabolism. Additionally, the pathways involved in membrane transport and metabolism of cofactors and vitamins were more abundant in the healthy partners. CONCLUSION: Our results suggested that the microbial composition might be affected in healthy partners cohabiting with UC patients, especially in terms of microbiota dysbiosis.

Seven-day triple therapy is a better choice for Helicobacter pylori eradication in regions with low antibiotic resistance.

AIM: To investigate whether 7-d triple therapies are still valid in populations with low levels of resistance. METHODS: A total of 1106 Helicobacter pylori (H. pylori)-positive patients were divided into three groups, each of which received one type of 7-d triple therapy. Therapeutic outcomes of the patients were assessed by the (13)C-urea breath test at 8 wk after treatment. The susceptibility of H. pylori to antibiotics was determined by an agar-dilution method. Data analysis was performed by χ(2) tests. RESULTS: The eradication rates in groups A, B and C were 90.71% (332/366), 90.46% (313/346) and 90.87% (189/208), respectively (P = 0.986). The resistance rates were 8.91% for clarithromycin, 14.78% for levofloxacin and 0% for amoxicillin. The eradication rate was significantly different between clarithromycin- and levofloxacin-resistant patients (P < 0.05) in group A. Patients whose treatment failed in group A also had a higher clarithromycin resistance rate than did successive patients (P = 0.034). However, levofloxacin resistance had no obvious influence on the eradication rate. Furthermore, three main antibiotics (clarithromycin, levofloxacin and amoxicillin) had lower DID (defined daily dose per 1000 inhabitants per day) in this city. CONCLUSION: Clarithromycin resistance is the main reason for the failure of 7-d triple therapy. In populations with low levels of resistance, a 7-d triple therapy is a viable choice. The choice of therapy should not be influenced by conditions in high antibiotic resistance regions.

Centralized isolation of Helicobacter pylori from multiple centers and transport condition influences.

AIM: To evaluate the efficacy of centralized culture and possible influencing factors. METHODS: From January 2010 to July 2012, 66452 patients with suspected Helicobacter pylori (H. pylori) infection from 26 hospitals in Zhejiang and Jiangsu Provinces in China underwent gastrointestinal endoscopy. Gastric mucosal biopsies were taken from the antrum for culture. These biopsies were transported under natural environmental temperature to the central laboratory in Hangzhou city and divided into three groups based on their transport time: 5, 24 and 48 h. The culture results were reported after 72 h and the positive culture rates were analyzed by a χ (2) test. An additional 5736 biopsies from H. pylori-positive patients (5646 rapid urease test-positive and 90 (14)C-urease breath test-positive) were also cultured for quality control in the central laboratory setting. RESULTS: The positive culture rate was 31.66% (21036/66452) for the patient samples and 71.72% (4114/5736) for the H. pylori-positive quality control specimens. In the 5 h transport group, the positive culture rate was 30.99% (3865/12471), and 32.84% (14960/45553) in the 24 h transport group. In contrast, the positive culture rate declined significantly in the 48 h transport group (26.25%; P < 0.001). During transportation, the average natural temperature increased from 4.67 to 29.14 °C, while the positive culture rate declined from 36.67% (1462/3987) to 24.12% (1799/7459). When the temperature exceeded 24 °C, the positive culture rate decreased significantly, especially in the 48 h transport group (23.17%). CONCLUSION: Transportation of specimens within 24 h and below 24 °C is reasonable and acceptable for centralized culture of multicenter H. pylori samples.

[Sequence analysis of the complete genome of bocavirus WLL-1].

Human bocavirus, which was firstly discovered in 2005, is a new human parvovirus associated with lower respiratory tract infection in children. In this study, a human bocavirus, named WLL-1 isolate, was identified in Wenlin County, Zhejiang Province. The genome of bocavirus WLL-1 has been sequenced and analyzed. Phylogenetic analyses showed that WLL-1 shares 99% homology with other bocaviruses recently reported, but also has some special variations.

Detection of let-7a microRNA by real-time PCR in gastric carcinoma.

AIM: To establish an accurate and rapid stem-loop reverse transcriptional real-time PCR (RT-PCR) method to quantify human let-7a miRNA in gastric cancer. METHODS: According to the sequence of let-7a miRNA, the stem-loop reverse transcriptional primer, the primers and quantitative MGB probes of real-time PCR were designed and synthesized. The dynamic range and the sensitivity of quantitative reverse transcriptional real-time PCR were determined. The levels of let-7a miRNA were examined in 32 gastric carcinoma samples by stem-loop RT-PCR method. RESULTS: The dynamic range and sensitivity of the let-7a miRNA quantification scheme were evaluated, the result showed the assay could precisely detect 10 copies of mature let-7a miRNA in as few as 0.05 ng of total RNA of gastric mucosa. The results of specificity analysis showed no fluorescence signal occurred even though 50 ng of human genomic DNA was added to the reverse transcription (RT) reaction. The expression level of let-7a miRNA in gastric tumor tissues was significantly lower compared to normal tissues in 14 samples from 32 patients. CONCLUSION: The stem-loop RT-PCR is a reliable method to detect let-7a miRNA which may play an important role in the development of gastric carcinoma.

Clinical evaluation of oligonucleotide microarrays for the detection of HBV mutants associated with lamivudine resistance.

OBJECTIVES: Long-term lamivudine administration in hepatitis B virus (HBV)-infected patients induces the emergence of HBV mutants with lamivudine resistance. The aim of the present study was to evaluate the clinical application of an oligonucleotide microarray in detecting HBV mutants associated with lamivudine resistance. METHODS: 947 HBV DNA-positive sera from: 388 patients receiving lamivudine treatment, 559 chronic hepatitis B patients not receiving lamivudine treatment, and 359 from HBV DNA-negative controls, were assayed for HBV mutations using the oligonucleotide microarray. Furthermore, follow-up studies were performed using 255 clinical samples from 51 patients treated with lamivudine at various periods. The results were compared with sequencing and real-time polymerase chain reaction (PCR). RESULTS: The HBV DNA polymerase Tyr-Met-Asp-Asp motif (YMDD) mutation was detected in all 388 samples containing lamivudine-resistant mutations identified by microarray. For the codons rt180, rt204 and rt207, the agreements between the microarray and sequencing data are 96.6, 98.5 and 100%, respectively. Two previously unreported mutants were also found in those samples. In the 947 samples collected from different patients, which were detected positive for HBV DNA by quantitative PCR, all but three weak-positive samples were positive by the microarray, demonstrating an agreement of 99.7%. In all the positive samples, mutations could be detected in the relevant loci of HBV DNA polymerase with lamivudine resistance. All of the 359 HBsAg-negative samples were shown to be negative for HBV DNA using the microarray method. Follow-up detection of the clinical samples from 51 patients treated with lamivudine demonstrated that the microarray method was able to detect mutations in mixed viruses that were infecting prior to sequencing. CONCLUSION: The oligonucleotide microarray can be conveniently utilized to detect mutant HBV in clinical serum samples.