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Pre-existing mutations in reverse transcription region of HBV in patients with different HBV infection stages

Yaojiang XU ; Yida YANG ; Yaodong ZHANG ; Yonggang CHEN ; Weiquan SONG

Chinese Journal of Clinical Infectious Diseases.2013;6(5):257-262. doi:10.3760/cma.j.issn.1674-2397.2013.05.001

Objective To compare the pre-existing mutations in reverse transcription region of HBV in patients with different HBV infection stages.Methods Totally 474 patients with chronic HBV infections,including 205 with chronic hepatitis B (CHB),153 with liver cirrhosis and 116 with hepatocellular carcinoma (HCC),were enrolled from the People' s Hospital of Shangyu and the First Affiliated Hospital of Zhejiang University during January 2011 and June 2013.All patients had not received nucleos (t)ide analogues treatment.HBV RT region mutations and genotypes were determined by PCR followed by sequencing.SPSS14.0 was used for statistical analysis.Results There were 387 (81.6％) patients with HBV genotype B,in which 156 were with CHB,124 were with liver cirrhosis,and 107 were with HCC.Nucleos(t)ide analogues-related mutations were observed in all the above 387 patients.rtS106C mutation was more popular in CHB and liver cirrhosis (14.1％ and 14.5％) patients than that in patients with HCC (4.7％) (x2 =6.126,6.207,P ＜0.05); And the positive rates of rtD134E/G/N/S mutations were also higher in CHB and cirrhotic patients (21.8％ and 20.2％) than that in HCC patients (10.3％,x2 =5.933,4.263,P ＜ 0.05).rtD134E/G/N/S and rtS106C mutations were correlated with HBeAg (P ＜0.01) and gender (P ＜ 0.05),but not with HBV virus load and age (P ＞ 0.05).The mutation frequencies in A-B interdomain were higher in CHB and cirrhotic patients (5.3％ and 5.6％) than that in HCC patients (3.5％,x2 =9.018,11.018,P ＜ 0.01).Conclusions Nucleos (t) ide analogues-related mutations exist in various HBV infection stages.rtSl06C and rtD134E/G/N/S mutations may be involved in necro-inflammation,and A-B interdomain mutations may be correlated with necro-inflammation,immune response and fibrosis in chronic liver diseases.

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Expression of interleukin-18 in liver tissue of patients with chronic hepatitis B and its clinical significance

Lei TANG ; Yufeng GAO ; Jinjin DAI ; Xu LI ; Guizhou ZOU

Chinese Journal of Clinical Infectious Diseases.2013;6(5):273-277. doi:10.3760/cma.j.issn.1674-2397.2013.05.004

Objective To investigate the expression of interleukin 18 (IL-18) in liver tissues of patients with chronic hepatitis B (CHB) and its clinical significance.Methods Totally 172 CHB patients admitted in the Second Affiliated Hospital of Anhui Medical University from January 2012 to December 2012 were enrolled.The tissue specimens were obtained by liver biopsy from all patients,and 22 normal liver specimens were also collected as controls.Liver function,serum markers of HBV and HBV DNA load were measured and liver inflammation grades and fibrosis stages were assessed.Immunohistochemical method was used to detect the expression of IL-18 in liver tissues.Spearman rank correlation was used to analyze the correlations between IL-18 expression and the above indicators.Results IL-18 was mainly expressed in cytoplasm of liver cells.It was positive or strong positive in liver cells of healthy people,but weakly positive in liver cells of CHB patients.The difference of IL-18 expression between CHB patients and healthy people was of statistical significance (x2 =42.124,P ＜ 0.05).In CHB patients,the expression of IL-18 was gradually enhanced with the increasing of ALT,inflammation grades and fibrosis stages (r =0.182,0.173 and 0.068,P ＜0.05),but it was not correlated with HBV DNA load and HBeAg (r =-0.05 and 0.041,P ＞0.05).Conclusion In CHB patients,the expression of IL-18 may be involved in liver injury and fibrosis formation.

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Effect of fimH gene on type 1 fimbriae adhesion of uropathogenic Escherichia coli: a preliminary study

Jianfei WANG ; Yang CAO ; Dianjun WEI

Chinese Journal of Clinical Infectious Diseases.2013;6(5):278-281. doi:10.3760/cma.j.issn.1674-2397.2013.05.005

Objective To investigate the effect of fimH gene on type 1 fimbriae adhesion of uropathogenic Escherichia coli (UPEC) and the gene variations between type 1 fimbriae adhesion positive and negative bacteria.Methods A total of 171 UPEC strains (not catheter associated) were collected from the Second Hospital of Tianjin Medical University,Tianjin First Center Hospital,and Tianjin Children's Hospital during January 2012 and January 2013.fimH gene was detected by PCR technique,and type 1 fimbriae adhesion was detected by yeast agglutination test.RT-PCR was used to exterminate gene transcript factor impacting on adhesion.Chi-square and Yates' chi-squared tests were performed to comparefimH gene sequences between the adhesion positive and negative bacteria.Results Among 171 UPEC strains,142 (83％) werefimH gene positive,and type 1 fimbriae was expressed in 98 strains (57％).All adhesion positive strains carriedfimH gene.Among 44 strains with positivefimH gene and negative adhesion RT-PCR revealed thatfimH gene did not transcript in 8 strains (18％).The sequencing results showed that gene mutation on the 51 st amino acid site was more prevalent in the adhesion positive group compared with the negative group (x2 =6.64,P =0.010).In adhesion,mutations on the 190th and 219th amino acid sites were observed in 6 strains and 7 strains of negative group,respectively; while not observed in the adhesion positive group (x2 =4.69 and 5.87,P ＜ 0.05).Negative adhesion in other 23 strains was not attributed to single nucleotide polymorphism.Conclusion Adhesion function of type 1 fimbriae mignt be affected by mutation and deletion offimH gene.Three key site mutations may also affect the adhesion function of type 1 fimbriae.Besides fimH gene,there may be other genes that can affect the adhesion function of type 1 fimbriae.

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Comparison of different susceptibility testing methods of tigecycline against Acinetobacter baumannii and Klebsiella pneumoniae

Cuie HE ; Yanchun LI ; Bin TIAN ; Hua YANG ; Zhidong HU

Chinese Journal of Clinical Infectious Diseases.2013;6(5):282-286. doi:10.3760/cma.j.issn.1674-2397.2013.05.006

Objective To compare different susceptibility testing methods of tigecycline against Acinetobacter baumannii and Klebsiella pneumoniae.Methods Fifty carbapenem-resietant A.baumannii (CRAB) strains and 49 K.pneumoniae strains were collected from Tianjin Medical University General Hospital during January and March 2012.Minimum inhibitory concentration (MIC) and inhibitory zone diameters for tigecycline were determined by broth microdilution,Vitek-2,MTS and disk diffusion methods.The results of Vitek-2,MTS and disk diffusion methods were compared with those of broth microdilution method.Results According to FDA standards,the susceptibilities of CRAB and K.pneumoniae to tigecycline determined by broth microdilution,Vitek-2 and MTS were 94.0％/91.8％,68.0％/91.8％ and 90.0％/91.8％,respectively.For CRAB isolates,the essential agreement (EA) and categorical agreement (CA) produced by Vitek-2 and MTS were 94.0％/72.0％ and 92.0％/90.0％.MICs determined by Vitek-2 were 1-2 dilutions higher than the reference method in 33 (66.0％) strains,and those determined by MTS were higher in 16 (32.0％) strains and lower in 11 (22.0％) strains.For K.pneumoniae isolates,the EA/CA produced by Vitek-2 and MTS were 95.9％/98.0％ and 83.7％/91.8％,respectively.MICs determined by Vitek-2 were 1-3 dilutions lower than the reference method in 17 (34.7％) strains,and those determined by MTS were 1-3 dilutions lower than the reference method in 39 (79.6％) strains.None of thetwo methods produced very major error (VME) and major error (ME) against two kinds of isolates.The results were determined by disk diffusion method using different breakpoints according different isolates.For CRAB,using ≥14 mm/≤ 10 mm as breakpoint,CA was 94.0％,which was higher than the breakpoint recommended by Jones et al (≥16 mm/≤12 mm,CA was 82.0％) ; and for K.pneumoniae,using the ≥ 14 mm/≤ 10 mm as breakpoint,CA was 93.9％,higher than the FDA Enterobacteriaceae breakpoint (≥ 19 mm/≤ 14 mm,CA was 67.3％).Conclusion For CRAB strains,MTS produces better consistence with broth microdilution,with several higher or lower MIC results.For K.pneumoniae strains,Vitek-2 has better correlation with reference method,with several lower MIC results.The consistence between disk diffusion method and broth microdilution is comparatively lower,and the breakpoint should be adjusted according to different bacteria.

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Comparison of shear wave velocity with noninvasive serum score systems in diagnosis of liver fibrosis of patients with chronic hepatitis

Wei CHEN ; Pengjian WENG ; Guosheng GAO ; Huadong YAN ; Dafeng MAO ; Chengliang ZHU

Chinese Journal of Clinical Infectious Diseases.2013;6(5):269-272. doi:10.3760/cma.j.issn.1674-2397.2013.05.003

Objective To evaluate the clinical application of liver shear wave velocity (SWV) in comparison with common serum score systems [AST to platelet ratio index (APRI),Forns,S index and FIB-4] in assessment of liver fibrosis in patients with chronic hepatitis.Methods A total of 237 chronic hepatitis patients with liver fibrosis confirmed by liver biopsy,who were admitted in Ningbo Second Hospital during October 2010 and April 2013,were enrolled in the study.Liver shear wave velocity were measured by acoustic radiation force impulse (ARFI),and the score of APRI,Forns,S index and FIB-4 were calculated based on the measurement of serum markers.Liver fibrosis stages were classified as S0-S4 according to the Scheuer scoring system,and stages ≥ S2 were identified as significant liver fibrosis.The diagnosis value of SWV,4 common score systems and their combination for significant liver fibrosis was evaluated by receiver operating characteristic curve (ROC).Results A significant linear correlation was found between SWV and the stage of fibrosis (r =0.46,P ＜ 0.01).The areas under the ROCs of SWV and 4 common score systems (APRI,Forns,S index and FIB-4) for the diagnosis of significant liver fibrosis were 0.758 (0.696-0.821),0.727 (0.662-0.793),0.777 (0.717-0.836),0.747 (0.684-0.810) and 0.737 (0.673-0.802),respectively.The area under the ROC of the combined prediction nodel established with Logistic regression was 0.810.Conclusion Liver shear wave velocity measured by ARFI is of clinical value in noninvasive assessment of liver fibrosis,and the prediction accuracy can be improved when it is combined with other noninvasive indices.

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Optimal therapy for patients with HBeAg positive chronic hepatitis B who have poor response to interferon α

Qi SHEN ; Zhan ZHANG ; Yong WU

Chinese Journal of Clinical Infectious Diseases.2014;7(1):45-48. doi:10.3760/cma.j.issn.1674-2397.2014.01.009

Objective To investigate the efficacy of interferon alpha (IFNα) plus adefovir dipivoxil (ADV) and tebivudine (LdT) monotherapy for patients with HBeAg positive chronic hepatitis B (CHB) who have poor response to IFNα treatment.Methods A total of 86 HBeAg positive CHB patients admitted to the Sixth People' s Hospital of Shaoxing during February 2010 and April 2013 were enrolled in the study.All the patients received IFNα monotherapy for 24 weeks and had poor responses.The patients were voluntarily divided into three groups:IFNα monotherapy group (n =21),IFNα plus ADV group (n =30) and LdT monotherapy group (n =35).Chi-square test was used to compare ALT normalization rates,rate of HBV DNA load ＜ 500 copies/mL,HBeAg seroconversion and HBsAg seroconversion rates among three groups.Results After 48 weeks of treatment,the ALT normalization rate in IFNα monotherapy group was 52.6％ (10/19),which was lower than those in IFNα plus ADV group (86.7％,26/30) and LdT monotherapy group (84.8％,28/33) (x2 =6.913 and 6.361,P ＜ 0.05).The rate of HBV DNA load ＜500 copies/mL in IFNα monotherapy group was 26.3％ (5/19),which was lower than those in IFNα plus ADV group (60.0％,18/30) and LdT monotherapy group (54.5％,18/33) (x2 =11.33 and 3.895,P ＜0.05).No HBeAg negative conversion or seroconversion was observed in IFNα monotherapy group,but it was observed in 6 (20.0％,6/30) patients in IFNαt plus ADV group and 7 (21.2％,7/33) patients in LdT monotherapy group (x2 =4.330 and 4.657,P ＜ 0.05).No HBsAg seroconversion was observed in three groups.There were no statistical significant differences in ALT normalization rates,rate of HBV DNA load ＜ 500 copies/mL,HBeAg seroconversion and HBsAg seroconversion rates between IFNαt plus ADV group and LdT monotherapy (x2 =0.042,0019 and 0.064,P ＞ 0.05).Conclusion For patients with HBeAg positive CHB who had poor response to IFNα treatment,IFNα plus ADV therapy and LdT monotherapy have the same efficacy in improvement of both liver function and virological response.

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Whole-genome sequence-based analysis of Klebsiella pneumoniae JM45

Jianming ZHU ; Rujin JIANG ; Xingbei WENG ; Kangle WU ; Haishen KONG

Chinese Journal of Clinical Infectious Diseases.2014;7(1):27-33. doi:10.3760/cma.j.issn.1674-2397.2014.01.006

Objective To investigate the distribution of β-lactamase genes in a pan-drug resistant Klebsiella pneumoniae isolate JM45.Methods Klebsiella pneumoniae JM45 was isolated from the blood sample of a patient admitted in the intensive care unit,the Second Affiliated Hospital,Zhejiang University School of Medicine on April 7,2010.The susceptibilities to 26 antibiotics were tested using E-test method.Cica-β-Test was performed to detect β-lactams,and modified Hodge test was performed to detect carbapenemase.Resistant genotypes were detected using PCR,DNA sequencing and BLAST algorithm.Whole genome sequencing (complete graph) was performed by high throughput Roche 454 sequencing approach to analyze the distribution of β-lactamase genes.Results Except polymyxin B and tigecycline,JM45 was resistant to other 24 kinds of antibiotics including cephalosporins and carbapenems.Several β-lactamases were positive in Cica-β-Test,and modified Hodge test was positive.Based on PCR typing,TEM-1,SHV-11,CTX-M-24 and VEB-3 were positive,but carbapenemase genes and metallo-β-lactamase genes were negative.A complete genome (chromosome) sequence (GenBank accession number:CP006656) and 2 plasmids sequences (GenBank accession number:CP006657,CP006658) were obtained by wholegenome sequencing.CTX-M-24 (Locus tag:N559_5233),TEM-1 (Locus tag:N559_5242) and VEB-3 (Locus tag:N559_5248) were positive in plasmid 1.CTX-M-24 located in insertion sequence (IS903-CTXM-24-ISEcp1),while TEM-1 and VEB-3 located in transposons (tnpA-TEM-1-rmtB and VEB-3-tnpA).SHV-11 (Locus tag:N559_2715) was positive in genome (chromosome),and 4 putative β-lactamase genes or β-lactamase domains were obtained:(1) metallo-β-lactamase domain protein (Locus tag:N559_0119,780 bp) ; (2) putative β-lactamase (Locus tag:N559_1633,1308 bp) ; (3) β-lactamase domain protein (Locus tag:N559_2279,813 bp); (4) β-lactamase domain protein (Locus tag:N559_3769,1101 bp).No insertion sequence or transposase gene was observed near SHV-11.Conclusion The resistance to antibiotics including cephalosporins and carbapenems is correlated with TEM-1,SHV-11,CTX-M-24,VEB-3 and 4 kinds of putative β-lactamase genes or β-lactamase domains.

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A novel evaluation system for T lymphocytes immune function based on the expression of costimulatory molecules in chronic HBV-infected patients

Lin WANG ; Chunnan ZHAO ; Songnan QI ; Qunxin PENG ; Jinfang SHI ; Guohao GU

Chinese Journal of Clinical Infectious Diseases.2014;7(1):53-59. doi:10.3760/cma.j.issn.1674-2397.2014.01.011

Objective To investigate the expression and significance of CD28,cytotoxic T-lymphocyte antigen-4 (CTLA-4),programmed death-1 (PD-1) and T cell immunoglobulin mucin-3 (Tim3) on T lymphocytes in chronic HBV-infected patients.Methods A total of 102 chronic HBV-infected patients,including 42 patients with chronic hepatitis B (CHB),30 patients with hepatitis B-induced liver cirrhosis (LC),and 30 patients with hepatocellular carcinoma (HCC),were enrolled from the First Affiliated Hospital to Soochow University during October 2012 and June 2013.Thirty healthy individuals were also enrolled as controls.Expression of CD28,CTLA-4,PD-1,Tim-3 on T lymphocytes in peripheral blood were determined by flow cytometry,and the differences among groups were analyzed using one-way ANOVA and LSD-t test.Spearman correlation test was performed to analyze the correlations of the expression of CD28,CTLA-4,PD-1,Tim-3 on T lymphocytes with HBV DNA loads,HBeAg and ALT.Results The expression of CD4 + CD28 +,CD8 + CD28 +,CD4 + CTLA-4 + in chronic HBV-infected patients were lower than those in healthy controls.CD4 + CD28 + expression in HCC group was lower than that in CHB group (t =2.373,P ＜ 0.05) ; CD8 + CD28 + expression in LC and HCC group was lower than that in CHB group (t =4.324 and 4.088,P ＜ 0.01) ; CD8 + PD-1 +,CD4 + Tim-3 + and CD8 + Tim-3 + expressions in CHB group were higher than those in LC,HCC group and healthy controls (t =3.051,3.130,3.121,3.254 and 3.723,P ＜0.01).CD8 + PD-1 + expression was positively correlated with ALT levels and HBV DNA loads (r =0.516 and 0.582,P ＜ 0.01) ; CD8 + Tim-3 + expression was also positively correlated with ALT levels andHBV DNA loads (r =0.578 and 0.556,P ＜0.01); PD-1 and Tim-3 expressions on CD8 T lymphocytes were positively correlated with each other (r =0.578,P ＜ 0.01).Conclusion The abnormal expression of the molecules on T lymphocytes in chronic HBV-infected patients is closely correlated with immune function disorder and the progression of the disease.

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Cerebrospinal fluid characteristics and clinical features in children with severe hand, foot and mouth disease induced by enterovirus 71 infection

Xinfeng ZHAO ; Yidong WU ; Yang GAO ; Lei ZHOU ; Shiyong ZHAO ; Yi WEI

Chinese Journal of Clinical Infectious Diseases.2014;7(1):60-63. doi:10.3760/cma.j.issn.1674-2397.2014.01.012

Objective To investigate cerebrospinal fluid characteristics and clinical features in children with severe hand,foot and mouth disease (HFMD) induced by enterovirus 71 (EV71) infection.Methods A total of 114 children with severe HFMD,in whom EV71 was detected by reverse transcription polymerase chain reaction (RT-PCR),were admitted in Hangzhou Children's Hospital during May and August 2013.Seventy-eight children with severe HFMD induced by other enteroviruses admitted at the same period served as controls.The results of cerebrospinal fluids (CSF) routine examination and biochemical tests,and the clinical symptoms were compared between two groups.Differences in enumeration data were compared with x2 test,and measurement data were compared with Mann-Whitney U test.Results The incidences of vomiting and limb shaking in EV71 infection group were 35.1％ and 50.9％,which were higher than those in control group (x2 =7.864 and 19.682,P ＜ 0.05).The incidence of limb shaking in children with nucleated cells count ≥ 100 × 106/L in EV71 group was higher than that with nucleated cells count ＜ 100 × 106/L (72.3％ vs.35.8％,x2 =14.740,P =0.000).The nucleated cells count,protein quantity and their positive rates in EVT1 infected group were higher than those in control group (Z =-9.458 and-6.591,P=0.000; x2=105.421 and 10.932,P =0.000 and 0.001).Conclusion The symptoms of nervous system damage and abnormal CSF examination were more serious in HFMD induced by EV71 infection,and in EV71 infected patients the incidence of limb shaking is correlated with nucleated cell count in CSF.

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Advances in antiviral therapy targeting to hepatitis C virus NS3/4A protease

Yan GUO ; Changye HUI ; Wei XIONG ; Wen ZHANG

Chinese Journal of Clinical Infectious Diseases.2014;7(1):89-93. doi:10.3760/cma.j.issn.1674-2397.2014.01.022

Hepatitis C virus (HCV) genome is of high variation,which results in persistent infection of HCV and increases the incidence of liver cirrhosis and hepatocellular carcinoma.Following the successful paradigm established for HIV protease inhibitors,HCV NS3-4A serine protease has been selected as the main target for the development of small molecule antiviral agents.In this article,we review recent progress in the discovery and development of HCV NS3-4A protease inhibitors,and discuss their antiviral activities,pharmacokinetic properties,side effects and resistance profiles.

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