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Objective@#To establish a high-throughput sequencing method for a whole genome in different hepatitis B virus (HBV) concentrations.@*Methods@#Two method of amplicon-sequencing and direct sequencing without PCR amplification were used for library construction in the three plasmids, including the low HBV load sample, the moderate HBV load sample and the high HBV load sample. Whole genome sequencing was performed on Illumina MiSeq platform.@*Results@#There are significant differences in data yield between the two different library construction method. Only a few reads could be mapped to the HBV genome for direct sequencing. However, three samples were successfully amplified by the nested PCR amplification and amplicon-sequencing showed that all HBV samples had a good coverage and depth, which was not affected by HBV concentration. The alignment rate of HBV genome approached 80%. A total of 27 intra-host single nucleotide variations (iSNVs) were identified and 13 iSNVs were low-frequency mutation in three samples. Compared with the high HBV load sample, mutations in the reverse transcription (RT) region was more easily appeared in the low HBV sample and the moderate HBV load sample.@*Conclusions@#Integrating nested PCR with high-throughput sequencing to the HBV whole genome sequencing is not only a practical method to detect the infection of HBV with a high-sensitivity and accuracy, but also enables to detect the mutation in the infected patient with low HBV copy number.
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Objective To analyze the drug resistance of Helicobacter pylori (Hp) strains isolated from teenagers (aged 13 to 19) in Xiaoshan area to metronidazole,clarithromycin,levofloxacin,amoxicillin and furazolidone. Methods A retrospective study was conducted on 1 452 teenagers who had endoscopic examination in the First People′s Hospital of Xiaoshan District from January 2012 to December 2017. Statis-tical analysis was performed to analyze the antibiotic resistance of Hp to various antibiotics. Results Teen-agers who were positive for Hp infection accounted for 16.46% (239). The resistance rates of Hp isolates to metronidazole and clarithromycin were 71.55% and 16.74% with an upward trend in the past two years. Among all Hp isolates, those resistant to levofloxacin, amoxicillin and furazolidone accounted for 5.02%, 0.0% and 0.84%,respectively. No significant change in the antibiotic resistance patterns of Hp was ob-served. There were 15.06% of Hp isolates resistant to both metronidazole and clarithromycin. No statistical-ly significant difference in antibiotic resistance patterns was found between Hp isolates from male and female teenagers in Xiaoshan area. Conclusion This study suggested that the Hp isolates showed high resistance to metronidazole,moderate resistance to clarithromycin and levofloxacin,and low resistance to amoxicillin and furazolidone. Attention should be paid to monitoring the antibiotic resistance patterns in teenagers and pro-moting individualized treatment with regard to the eradication strategy of Hp infection.
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Objective To evaluate combined sampling at multiple sites of gastric mucosa for Helicobacter pylori (HP) culture.Methods A total of 258 patients with upper gastrointestinal symptoms received 13C-urea breath test between August 2014 and May 2015.During endoscopy,gastric mucosa biopsy samples from the lesser curvature of the antrum (A),the greater curvature of the antrum (B),gastric angle (C) and the body of the stomach (D) were collected to isolate HP strains.The positive rates of HP based on combined sampling and single site sampling were compared with a Nemenyi test.Results Consistency between 13C-urea breath test and HP culture was 82.56％.There was significant difference between the single site sampling and two-site sampling in the positive rate of HP,except for the body of the stomach (P＜0.05).There was significant difference in the positive rate of HP between the single site sampling and three-site sampling (P ＜ 0.05).There was no significant difference between any two-site sampling in the lesser curvature of the antrum and the body of the stomach,gastric angle and the body of the stomach,the greater curvature of the antrum and the body of the stomach,and any three-site sampling (P＞0.05).Conclusion The combined sampling of the lesser curvature of the antrum and the body of the stomach have the highest cost-effectiveness in HP culture compared with the single site sampling and three-site sampling.
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Objective To investigate the in vitro quinolones resistance in Helicobacter pylori ( HP) strains isolated in Zhejiang province and to provide references for the clinical application of quinolones in the eradication of HP infection.Methods A total of 713 HP strains were isolated from several hospitals located in different regions of Zhejiang province from April to June 2013.Drug sensitivity test was performed to measure the resistance of those HP strains to levofloxacin and moxifloxacin by using the agar dilution method recommended by the Clinical and Laboratory Standards Institute ( CLSI) .The antibiotic solutions were added to the agar and diluted into corresponding critical concentrations.The mixed liquors were placed in dilution plates and added with the bacteria suspension.If there were bacterial expansion on the plates after incuba-tion, the strains were considered to be drug-resistant strains.Results The resistance rates of 713 HP strains to levofloxacin and moxifloxacin were 19.50%.In addition to the Jinhua area, drug resistance rates in the other 7 areas including Hangzhou, Wenzhou, Shaoxing, Jiaxing, Taizhou, Lishui and Zhoushan were above 15%.The highest drug resistance rate of HP was found in Wenzhou, which was significantly higher than that in Jinhua area (26.40%vs 9.52%, P<0.05).No significant differences in the resistance rate of HP to quinolone were found between Wenzhou area and the other regions in Zhejiang.Conclusion Com-pared with amoxicillin and furazolidone, higher resistance rates of HP to levofloxacin and moxifloxacin were observed in Zhejiang province.Because the mechanisms of drug resistance to levofloxacin and moxifloxacin were same and the drug targets within HP to which the two antibiotics binding were similar, levofloxacin and moxifloxacin shared common characteristics of drug resistance among HP strains, indicating that prescription of the two antibiotics in replacement should be avoided in clinical treatment of HP drug-resistant strains.
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Objective To study the infection status of Helicobacter pylori (H. pylori) and sensitivity to commonly used antibiotics in Taizhou district,Zhejiang province. Methods 39 099 cases aged between 5 and 95 years old(mean as 48.42 years)were involved during January 2010 to December,2013 for this study. Sex ratio was 1 ∶ 0.95. Yearly distribution of the number of cases were 5 031,6 709,11 902 and 15 457 in 2010,2011,2012 and 2013,respectively. Gastric mucosal specimens were collected and H. pylori strains were isolated and cultured in the same platform in Zhiyuan Medical Inspection Institute of Hangzhou. Resistance tests of all the H. pylori isolates were performed to 6 commonly used antibiotics:metronidazole,clarithromycin,amoxicillin,gentamicin, levofloxacin and furazolidone with the agar dilution method. The antibiotic resistance rates of H. pylori strains isolated during year 2010-2013 and the changing trends were analyzed. Results Resistance rates to levofloxacin and clarithromycin kept at higher level and the highest was in 2011 and then decreased in both 2012 and 2013 (P<0.01). The resistance rates to both levofloxacin and clarithromycin reached the highest in 2011(P<0.01),and decreased thereafter,with no significant change in 2013 to 2012(P>0.05). Conclusion Antibiotic resistance rate against metronidazole for HP isolate was highest. Resistance rate against amoxicillin and furazolidone,gentamicin was low. Clinical treatment should choose amoxicillin and furazolidone,gentamicin. The resistance rates to levofloxacin and clarithromycin had been seen at a significantly downwarding trend since 2011. However,the combined resistance rates to levofloxacin and clarithromycin did not seem to reduce since 2012.
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Objective To study the infection status of Helicobacter pylori (H. pylori) and sensitivity to commonly used antibiotics in Taizhou district,Zhejiang province. Methods 39 099 cases aged between 5 and 95 years old(mean as 48.42 years)were involved during January 2010 to December,2013 for this study. Sex ratio was 1 ∶ 0.95. Yearly distribution of the number of cases were 5 031,6 709,11 902 and 15 457 in 2010,2011,2012 and 2013,respectively. Gastric mucosal specimens were collected and H. pylori strains were isolated and cultured in the same platform in Zhiyuan Medical Inspection Institute of Hangzhou. Resistance tests of all the H. pylori isolates were performed to 6 commonly used antibiotics:metronidazole,clarithromycin,amoxicillin,gentamicin, levofloxacin and furazolidone with the agar dilution method. The antibiotic resistance rates of H. pylori strains isolated during year 2010-2013 and the changing trends were analyzed. Results Resistance rates to levofloxacin and clarithromycin kept at higher level and the highest was in 2011 and then decreased in both 2012 and 2013 (P<0.01). The resistance rates to both levofloxacin and clarithromycin reached the highest in 2011(P<0.01),and decreased thereafter,with no significant change in 2013 to 2012(P>0.05). Conclusion Antibiotic resistance rate against metronidazole for HP isolate was highest. Resistance rate against amoxicillin and furazolidone,gentamicin was low. Clinical treatment should choose amoxicillin and furazolidone,gentamicin. The resistance rates to levofloxacin and clarithromycin had been seen at a significantly downwarding trend since 2011. However,the combined resistance rates to levofloxacin and clarithromycin did not seem to reduce since 2012.
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<p><b>OBJECTIVE</b>To study the infection status of Helicobacter pylori (H. pylori) and sensitivity to commonly used antibiotics in Taizhou district,Zhejiang province.</p><p><b>METHODS</b>39 099 cases aged between 5 and 95 years old (mean as 48.42 years) were involved during January 2010 to December, 2013 for this study. Sex ratio was 1 : 0.95. Yearly distribution of the number of cases were 5 031, 6 709, 11 902 and 15 457 in 2010, 2011, 2012 and 2013, respectively. Gastric mucosal specimens were collected and H. pylori strains were isolated and cultured in the same platform in Zhiyuan Medical Inspection Institute of Hangzhou. Resistance tests of all the H. pylori isolates were performed to 6 commonly used antibiotics:metronidazole, clarithromycin, amoxicillin, gentamicin, levofloxacin and furazolidone with the agar dilution method. The antibiotic resistance rates of H. pylori strains isolated during year 2010-2013 and the changing trends were analyzed.</p><p><b>RESULTS</b>Resistance rates to levofloxacin and clarithromycin kept at higher level and the highest was in 2011 and then decreased in both 2012 and 2013 (P < 0.01). The resistance rates to both levofloxacin and clarithromycin reached the highest in 2011 (P < 0.01), and decreased thereafter, with no significant change in 2013 to 2012 (P > 0.05).</p><p><b>CONCLUSION</b>Antibiotic resistance rate against metronidazole for HP isolate was highest. Resistance rate against amoxicillin and furazolidone, gentamicin was low. Clinical treatment should choose amoxicillin and furazolidone, gentamicin. The resistance rates to levofloxacin and clarithromycin had been seen at a significantly downward trend since 2011. However, the combined resistance rates to levofloxacin and clarithromycin did not seem to reduce since 2012.</p>
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Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Humanos , Pessoa de Meia-Idade , Adulto Jovem , Antibacterianos , Farmacologia , Farmacorresistência Bacteriana Múltipla , Helicobacter pyloriRESUMO
Objective To compare disk diffusion with E-test methods for clarithromycin susceptibility testing of Helicobacter pylori (H.pylori).Methods A total of 44 strains of H.pylori were isolated from gastric mucosa biopsy from patients undergoing gastroscopic examination.Disk diffusion and E-test methods were used for clarithromycin susceptibility testing of H.pylori.The agreement of disk diffusion and E-test was assessed by linear regression analysis.Results The minimum inhibitory concentration (MIC) tested by E-test method ranged from 0.016 to 256 μg/mL,and drug resistance was observed in 12(27.3％) isolates.In range of 0-35 mm of inhibition diameter,the results of disk diffusion method were correlated well with the MICs obtained by E-test method (r2 =0.91,P ＜0.01).Regression analysis showed that with inhibition diameters≥ 18 mm as considered sensitive to clarithromycin and ≤ 15 mm as resistant,the agreement was 100％ between two methods.Conclusion The disk diffusion method is equivalent to the E-test method for clarithromycin susceptibility testing of H.pylori,which can be an alternative method for clinical application.
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Objective To investigate condition of single drug or multiple drug resistance and sensitivities of different combinations of antibiotics in Helicobacter pylori (H.pylori) infected patients of different ages in Jiaxing City,Zhejiang Province.Methods From January 2007 to December 2011,a total of 6280 patients underwent gastroendoscopy examination were enrolled,the average age of them was 50.8 years old.Among them,129 cases were less than 20 years old,1802 cases were between 20 and 40,3016 cases were between 40 and 60,and 1333 cases were over 60.The mucosa of gastric antrum was collected for H.pylori culture.Drug sensitivity of isolated H.pylori strains was tested with metronidazole,amoxicillin and gentamycin,furazolidone,clarithromycin and levofloxacin.x2 test was performed for the comparison of drug resistance among different age groups.Results Among 6280 specimens,H.pylori culture of 2311 cases was positive (positive rate 36.80％).The positive rate of patients aged between 40 and 60 was the highest (38.43％,1159/3016),and that of patients over 60 was the lowest (33.76％,450/1333).The drug resistance rate of metronidazole in patients between 20 and 40 years reached 98.02％ (644/657) and was the highest.The drug resistance rate of clarithromycin in patents over 60 years old was the highest,which was 22.67％(102/450).The drug resistance rates of the levofloxacin,furazolidone,amoxicillin and gentamicin were low of all ages.Mixed resistance of two antibiotic appeared in 586 strains,mixed resistance rate of metronidazole and clarithromycin was the highest (259 strains,15.23 ％).Mixed resistance of three drugs appeared in 49 strains,mixed resistance rate of metronidazole,clarithromycin and levofloxacin was the highest (32 strains,12.88％).A total of 1691 strains were sensitive to the combination of amoxicillin and gentamycin,the sensitive was rate 99.41％.The sensitive rate of the combination of amoxicillin and furazolidone,gentamycin and furazolidone was both 94.24％.Conclusions Metronidazole presented high drug resistance of all ages,and should not be chosen.The combination use of amoxicillin and gentamicin is the ideal antibiotic combination for different ages.The combination of gentamycin and furazolidone is recommended for patients who are allergic to penicillin.
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Objectives To characterize the assay method and the clinical symptoms of the infant infection of the human bocavirus in order to advance the diagnositic and therapeutic technology for human bocavirus. Methods The DNA and specific antibody of human bocavirus from pharyngeal extracts, nasopharyngeal swab and serum were detected using real time PCR and Western blot. The overall diagnosis and analysis of the clinical symptoms, sign and conventional experimental test were performed, and meanwhile the long-term follow-up was done. Results 7 specimens were detected as human bocavirus positive from 240 pharyngeal extracts (7/240), and the positive ratio was 2.92%. The clinical symptoms were mainly high fever and temporary cough. and the experimental index of peripheral blood leucocyte, C-reaction protein, blood sedimentation and hemobiochemistry were usually in normal region. No specific characteristic was observed by the thoracic X-ray. and cardio-pulmonary function was normal through the long-term investigation. Conclusions Human bocavirus was one of pathogens inducing children lower respiratory tract infections. The real time PCR and Western blot were sensitive and specific detection technologies.
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Objective To investigate the the distribution of Helicobacter pylori (H. pylori) vacA gentypes and the relationship between the presence of specific genotypes and clinical diseases in Zhejiang of China. Methods 262 Helicobacter pylori strains were colleted from 8 districts of Zhejiang,chromosome DNA was extracted and polymerase chain reaction (PCR) was carried out to determine the polymorphism of vacA and cagA with specific primers.PCR results were analyzed statistically according to their isolated original and clinical outcomes. Results VacA m1b, vacAm2 and vacAm1bm2 were found positive in 27.10%,4.89% and 4.20% of the 262 H.Pylori strains respectively. There was no significant difference in vacA genotypes among different districts of Zhejiang. 26.47%(27/102)of vacA m1b,66.67%(68/102) vacAm2 and 2.94%(3/102)vacAm1bm2 Helicobacter pylori strains were isolated from chronic gastritis; 29.41%(40/136)of vacA m1b,61.76%(84/136)vacAm2 and 3.68%(5/136)vacAm1bm2 Helicobacter pylori strains were isolated from Peptic Ulcer;and 16.67%(4/24)of vacA m1b,75.00%(18/24) vacAm2 and 4.17%(1/24) vacAm1bm2 Helicobacter pylori strains were isolated from gastric cancer; There was no significant difference in vacA genotypes among different clinical disease. Conclusion CagA+ and vacAs1/ m2 are predominant genotypes of H. Pylori in 8 districts of Zhejiang province. However, the relationship between vacA genotypes of H. pylori and the clinical disease can be identified in this study.
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Objective To establish a oligochip method for detection of HBV Lamivudine drug resistance and evaluate the clinical role of the chip system.Methods 388 HBV DNA positive sera from patients receiving lamivudine treatment and 559 chronic hepatitis B patients not receiving lamivudine treatment,and 359 sera from HBV DNA negative controls were assayed for HBV mutations utilizing oligonucleotide microarray.Meanwhile,these results were contrasted with Quantitative PCR and DNA sequencing method.Results The results of clinical evaluation shows that for the codons 528,552 and 555,the agreements between the microarray and sequencing data are 96.6%,98.5% and 100%,respectively.In the 559 samples,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%.All the 359 HBsAg negative samples were shown to be negative for HBV DNA by the microarray method.Conclusions The HBV-Lamivudine oligochip is eligible to detecting wild type HBV and HBV lamivudine-mutants in patient's sera.It has great potential application of administration for lamivudine treatment in HBV patients.