Objective To study Kaposi's sarcoma-associated herpesvirus(KSHV)infection in chronic hepatitis B(CHB)patients and its correlation with hepatitis B virus(HBV)replication and treatment-related factors.MethodsEnzyme-linked immunosorbent assay(ELISA)with recombination protein KSHV ORF65 was employed to detect the KSHV antibody and real-time polymerase chain reaction(PCR)was performed to detect KSHV DNA and HBV DNA in CHB patients.Age,HBV replication and licorice preparation treatment of patients were further analyzed.Comparison of rates was done using X~2 test.Results KSHV ORF65 antibody positive rates were 27.3% in 161 male CHB patients and 30.0% in 50 female patients(X~2=0.135,P＞0.05).The KSHV infection rates were increased with age,but this tendency was not obvious in patients older than 40 years old.The highest infection rate was in age group of 31-40 years old which was 37.1%.The positive rate of HBV DNA in CHB patients with KSHV infection was 73.5%,which was 56.3% in uninfected patients(X~2=3.969,P＜0.05).The average plasma level of KSHV DNA in patients treated with licorice preparations was 204.7 copy/mL and that in patients without licorice preparation treatment was 533.9 copy/mL.Eight patients were KSHV DNA positive(KSHV DNA＞ 100 copy/mL)in 16 patients treated with licorice preparations and 23 were positive in 33 patients without licorice preparation treatment.Conclusions The KSHV infection rates are increased with age of CHB patients.KSHV infection may interfere with HBV replication and licorice preparations may suppresss KSHV replication in vivo.

Objective:To construct NMB0315 eukaryotic expression recombinant vector ,detect specific humoral and cellular immune response induced by the recombint DNA vaccine intramuscularly in female BALB /c mice,evaluate the immunocompetence and immunoprotection of the vaccine , so as to provide experimental basis for the development of a novel nucleic acid vaccine against N.meningitidis serogroup B .Methods: The whole NMB0315 gene was amplified by PCR from the standard strains MC 58 genomic DNA,cloned into a plasmid pcDNA3.1(+),identified by double digestion of the recombinant plasmid with restriction enzymes and se -quencing.The recombinant vector pcDNA 3.1 (+)/NMB0315 was transfected into eukaryotic COS-7 cells and RAW264.7 cells, the NMB0315 protein was detected by immunocytochemical method and Western blot respectively .The levels of specific humoral and cellular immune response were detected after inoculating in female BALB /c mice intramuscularly with the recombinant plasmid .The immune protective effect was investigated with the DNA vaccine and the bactericidal titer of the immune serum was deter mined by serum bactericidal assay ( SBA ) in vitro.Results: The recombinant pcDNA3.1 (+)/NMB0315 was effectively transcripted and expressed in eukaryotic cells and the specific humoral and cellular immune responses were induced in the inoculated mice .In the re-combinant pcDNA3.1(+)/NMB0315 group ,the levels of serum IgG,IgG1,IgG2a,IgG2b and IgG3 and genital tract sIgA were significantly higher than in controls ( P<0.001 ) .The stimulation index in the culture supernatant of the spleen lymphocytes of the vaccine group was higher than that of the control group (P<0.05).The ratios of serum IgG2a/IgG1 in the DNA vaccine group were less than 1.The bactericidal titer of the NMB 0315+CpG group reached 1:128 following three immunizations , the protection rate of the vaccine group was 70%against the N.meningitidis strain MC58.Conclusion:The NMB0315 nucleic acid vaccine could induce higher levels of humoral immunity and cellular immunity and showed effective protection against N .meningitidis serogroup B , the immune serum had strong bactericidal activity in vitro .

Objective To investigate the specific humoral immune response and cellular immune response induced by DNA vaccine with Neisseria gonorrhoeae porin B (PorB) fused with B subunit of Escherichia coli heat-labile enterotoxin B (LTB) in mice. Methods Target genes of porB, ltB and ltB-porB were amplified by polymerase chain reaction (PCR) and cloned into eukaryotic vector pcDNA3.1(-). The recombinants were identified by PCR, enzyme digestion and DNA sequencing.The vectors were transfected into Hela cells, and expressed proteins were checked by cytoimmunofluorescence. Female BALB/c mice were intranasally immunized with recombination vectors. The humoral immune response and cellular immune response were detected by enzyme linked immunosorbent assay (ELISA) and methyl thiazolyl tetrazolium (MTT) colorimetric assay. The expressions of recombination vectors in intranasal mucosal tissues of the immunized mice were detected by immunohistochemistry. The means between groups were compared by analysis of variance. Results All the three recombinants were expressed in Hela cells and intranasal mucosal tissues. The PorB specific IgG in serum and sIgA in vaginal secretions in DNA vaccine immunized mice were significantly higher than those in controls (P＜0.01 ; P＜0.05). Moreover, the sIgA level in pcDNA3.1 (-)/ltB-porB group was higher than that in peDNA3, 1(-)/porB group (P＝0. 002). The levels of interferon-gamma (IFN-γ) and interleukin-4 (IL-4) in the supernatants and stimulation index (SI) of spleen lymphocyte culture in pcDNA3, 1(-)/porB group were (170.04±23.89) pg/mL, (114.68±14.27) pg/mL and 1. 68±0.19, respectively; and those in pcDNA3, 1(-)/ltB-porB group were (161.42±27.50) pg/mL, (124.16±19.04) pg/mL and 1.73±0.28, respectively; which were both higher than those in pcDNA3.1(-)/ phosphate buffered saliae (PBS) group (P＜0. 01; P＜0.05) and pcDNA3.1 (-)/ltB group (all P＜0.05), while there was no significant difference between pcDNA3.1 (-)/ltB-porB group and pcDNA3. 1 (-)/porB group (0. 998, 0. 696, 0. 994; all P＞0.05). Conclusions The constructed DNA vaccines are all successfully expressed in Hela cells and murine intranasal mucosal tissues. The mucosal immunization of the vaccines [pcDNA3. 1 (- )/porB and pcDNA3.1 ( -)/ltBporB] could induce humoral immune response and cellular immune response, especially mucosal immune response. It is confirmed that mucosal adjuvant LTB could promote PorB to induce higher level of mucosal immune response in mice.

Objective To study the uncertainty and traceability of HBV DNA assays and discuss the comparability of results among different detection systems. Methods Different detecting systems were used to detect HBV DNA using the national standard substance as "quality control substance". The uncertainty of the results was evaluated referring "Guidelines for estimating and reporting measurement uncerTAinty of chemical test results" of NATA The results were traced back to the national standard substance. According to the CLSI document EP9-A2, the results were analyzed and subjected to bias estimation with the t(0.05sv) √u2b1+ u2b2 as the criterion clinically accepted to investigate the comparability of different detecting systems. Results The means (-y) measured by 3 HBV DNA assay systems were 6.15,5.88,and 6.31 lg(kIU/L) respectively. Except system A,both the biases of system B and C had statistical significance (all P ＜ 0. 05) and expanded uncertainty of three detection systems was varied, but the difference was within the maximum acceptable range (± 0. 5) of the external quality assessment by National Center for Clinical Laboratory. Being traceable to national standard substance, the results of HBV DNA of the three detecting systems were (5.45 ± 1.23), (5.55 ± 1.32) and (5.42 ± 1.25) lg(kIU/L), respectively.There was significant difference among three systems (F = 5.63, P ＜ 0. 05). Comparing system A and B,there was significant difference in statistic (q = 5. 12, P ＜ 0. 05) and the difference between system B and C also had statistically significant (q = 6. 85, P ＜ 0. 05), but the results between system A and C had no statistical difference (q = 1.85,P ＞ 0. 05). Among these three systems, the difference of any two detection systems had no statistical significance (all P ＞ 0. 05). It showed that system bias was acceptable in clinical application and the results between different systems were comparable. Conclusions It is necessary to estimate the uncertainty and traceability when comparing the HBV DNA assay among the different labs. It also needs to estimate the bias of different systems and evaluate the clinical acceptability to ensure the accuracy and comparability of the results.

Objective To investigate antimicrobial resistance among gram-positive cocci in China in 2008.Methods From June 2008 to December 2008,1171 consecutive and non-repetitive gram-positive cocci were collected from 12 teaching hospitals.The MICs of antibacterial agents was determined by agar dilution method.Results The prevalence of MRSA and methicillin-resistant coagulase-negative Staphylococci(MRSCoN) was 49.9%(232/465) and 74.0%(179/242),respectively.The MRSA prevalence ranged from 33.3% to 65% in different regions.About 71.1%(108/152) of Staphylococcus aureus from respiratory tract specimens,48.3%(28/58) of Staphylococcus aureus from blood samples,and 36%(68/189) of Staphylococcus aureus from the pus,wound and sterile body fluid samples were resistant to methicillin.The susceptible rates of MRSA to trimethoprim/sulfamethoxazole(SXT) and chloramphenicol were 81.5%(183/232) and 89.7%(208/232).Susceptibility to gentamicin, erythromycin, clindamycin, tetracyclines,rifampicin,and quinolones were from 3.9% to 35.0%.All Staphylococci isolates were susceptible to vancomycin,teicoplanin,and linezolid.Three vacomycin-resistant Enterococcus faecium strains were found in this study.About 96.2%(101/105) of Enterococcus faecalis and 97%(130/134) of Enterococcus faecium were susceptible to linezoild.Fifty-one out of 105 of Enterococcus faecalis(48.6%)and 101 out of 134 Enterococcus faecium(75.4%)were resistant to high concentration gentaroicin.The susceptibility of Enterococcus faecalis to all the antibiotics except for chloramphenicol and tetracycline was higher than that of Enterococcus faecium.Enterococcus faecium isolates showed a high resistant prevalence to most of antibiotics except glycopeptides and linezolid.The prevalence of PISP among 225 isolates was was 36.6%(15/41),and the prevalence of PNSSP from the other patients ranged from 15.4% to 26.6%.The susceptible rates of PSSP to cefprozil,cefuroxime and cefaclor were 67.5%(114/169),66.3%(112/169) and 61.5%(104/169),respectively.All the PISP isolates were resistant to the above three antibiotics.Teicoplanin,vancomycin and linezolid were the most active agents against Staphylococcus pneumoniae(susceptible rate,100%).About 96.9%,97.8% and 98.2% Staphylococcus pneumoniae isolates were susceptible to gatifloxacin,levofloxacin,and moxifloxacin,respectively.The susceptible rates of Staphylococcus pneumoniae to ceftriaxone,chloramphenicol and amoxicillin/clavulanic acid were 81.3%,77.3%,and 68.0%,respectively.The susceptibility of Staphylococci pneumoniae to macrolides,SXT and tetracycline ranged from 11.6% to 23.6%.Conclusions The prevalence of VRE is low in China.However,methicillin-resistance among Staphylococci isolates was high.The prevalence of PNNSP isolated from (≤)3 years children is higher than in the other age population.Teicoplanin,vancomycin,and linezolid remain high activity against Staphylococci,Enterococcus faecalis,Enterococcus faecium,and Staphylococcus pneumoniae.

Objective To investigate antimicrobial resistance among gram-positive cocci in China in 2009. Methods From June to December 2009, 1169 consecutive and non-repetitive gram-positive cocci were collected from 12 teaching hospitals at 9 cities. The minimal inhibitory concentration (MIC) of antibacterial agents was determined by agar dilution method. Results The prevalences of methicillinresistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase-negative Staphylococci (MRCoNS) were 45.3% (211/466) and 89. 5% (214/239), respectively. The isolation rate of MRSA was 33. 3%-68. 1% from different samples. All Staphylococci isolates were susceptible to vacomycin, teicoplanin and linezolid. Five point five percent (7/128) E. faecium strains were resistant to vacomycin. All E.faecalis strains were susceptible to vacomycin. About 99. 1% (108/109) of E. faecalis and E. faecium were susceptible to linezoild. The prevalence of penicillin-intermediate Streptococcus pneumoniae (PISP) was 21.6% (48/222). Only 1 (0. 5%, 1/222) Streptococcus pneumoniae strain was resistant to penicillin.Teicoplanin, vancomycin, linezolid and tigecycline were the most active agents against Streptococcus pneumoniae (susceptible rate 100% ). Conclusions The high prevalence of methicillin-resistance is among Staphylococcus strains. Different samples show a different MRSA prevalence. Teicoplanin, vancomycin and linezolid show very high activity to Staphylococci,E. faecalis, E. faecium and Streptococcus pneumoniae.

Objective To investigate antimicrobial resistance among gram-positive cocci in China in 2006.Methods From Jun 2006 to Dec 2006,674 consecutive and non-repetitive gram-positive bacteria were collected from 7 teaching hospitals.The minimal inhibitory concentration(MICs)of antibacterial agents were determined by agar dilution method.Results The prevalence of penicillin.resistant(ease)and pemcllhn. intermediate S.pneumoniae(PISP)among 100 isolates was l%and 19%,respectively.Teicoplanin and vancomycin were the most active agents against S.pneumoniae.97% and 98% S.pneumoniae isolates were susceptible to levofloxacin and moxifloxacin,respectively.The susceptibility of amoxicillin/clavulanic acid, ceftriaxone and chloramphenicol are 96%,87% and 73%,respectively.The susceptible rates of penicillin. susceptible S.pneumoniae(PSSP)to cefprozil and cefaclor were 62% and 55.7%,respectively.All the PISP and PRSP isolates were resistance to the two antibiotics.The susceptibility to macrolides,trimethoprim/ sulfamethoxazole and tetracycline was lower than 35%.The prevalence of methicillin-resistant Staphylococcus aureus(MRSA)and methicillin.resistant coagulase-negative Staphylococci(MRSCON)was 48%(33%-84%)and 81%(69%-94%),respectively.The susceptible rates of MRSA to trimethoprim/ sulfamethoxazole,chloramphenicol,rifampin,and the other antibiotics in this study were 72%,66%and 45%,respectively.The susceptible rate of MRSA to marcrolides,aminoglycosides,tetracyclines and quinolones were not more than 18%.56%(30%-86%)of E.faecalis and 80%(50%.100%)of E.faecium were highly resistant to gentamicin.The susceptibility of E.faecalis to all the antibiotics except chloramphenicol and tetracycline were higher than E.faecium.All isolates of S.aureus,CoNS and E.faecalis tested were susceptible to vacomycin and teicoplanin.There were two vacomycin.resistant E.faecium strains isolated from Hangzhou.Conclusions Antimicrobial resistance patterns of gram.positive cocci differed in different regions.The resistance of gram-positive cocci to the antibiotics in this study this year was a little higher than the data of the year of 2005.Teicoplanin and vancomycin remained very high activity to gram-positive cocci.

Objective To explore the immune mechanism of negative results of immune tests of schistosomiasis japonica pa?tients. Methods Totally 142 schistosomiasis patients(positive stool examinations)of Poyang Lake region were tested by ELI?SA method,and the ROC curve was applied to determine the high and low response of the patients. The levels of cellular immu?nity and cytokines of high and low responders were compared. Results Totally eight schistosomiasis patients were found as low responders. Besides SWAP?IgA(t= -1.588,P > 0.1),the levels of isotype antibodies were significantly lower in the low re?sponders compared with those in the high responders(t = -14.517 to -2.866,all P < 0.05). In the low responders,the propor?tion of CD3+T was increased;and the proportions of CD4+T,CD8+T,CD4+CD25+Treg,and the ratio of CD4+/CD8+ were all de?creased,but all of them were not significant(t = -1.72 to 0.974,all P > 0.05)compared with those in the high responders. The differences of IFN?γ and IL?10 between the high and low responders were both not significant(t= -2.426 to 0.216,all P >0.05). Conclusions There is a significant difference between the high and low responders only in the levels of isotype antibod?ies. One of the reasons of low response in the immune tests is the much lower antibody level after the antigen?antibody compound is completely formulated.

Objective Toinvestigateantimicrobialresistanceamonggram-positivecocciinChinain 2013.Methods Retrospectivestudy.FromJune2013toDecember2013,1663consecutiveandnon-repetitive gram-positive cocci were collected from 15 teaching hospitals. The minimal inhibitory concentration ( MIC) of antibacterial agents was determined by agar dilution method. A retrospective study was conducted on rates of resistance to antimicrobial agents. The prevalence of penicillin-resistant Streptococcus pneumoniae ( PRSP) between children and adult patients and the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) between elder group and younger adult patients were compared using chi-square test. Results The prevalence of PRSP in children below 3 years old ( 72. 9%, 51/70 ) was higher than adult patients (55. 2%, 106/192) (χ2 =6. 653,P<0. 05). About 94. 9%(261/275) and (92. 7%,255/275) of S. pneumonia were resistant to erythromycin and clindamycin. All S. pneumoniae strains were susceptible to teicoplanin, vancomycin, linezolid, tigecycline and daptomycin. Penicillin still showed very high activity against Streptococcus spp. β-Hemolytic group. More than 60% of Streptococcus spp.β-Hemolytic group were resistant to erythromycin, clindamycin and tetracyclines. The prevalence of MRSA and methicillin-resistant coagulase-negative Staphylococci(MRCoNS) was 39. 7%(229/576) and 80. 6%(224/278), respectively. The MRSA prevalence ranged from 24. 2% to 70. 0% in different regions. About 52. 6%( 100/190 ) of Staphylococcus aureus from respiratory tract specimens, 38. 5%(40/104)of Staphylococcus aureus from blood samples, and 29. 7%(58/195) of Staphylococcus aureus from wound and pus were resistant to methicillin. The prevalence of MRSA in elder group ( 48. 6%, 84/173 ) was higher than that in younger adult patients (35. 7%, 144/403)(χ2 =8. 322,P <0. 05). The susceptibility rates of MRSA to chloramphenicol and trimethoprim/sulfamethoxazole were 86. 4% ( 244/228 ) and 94. 7% ( 237/228 ) , respectively. Susceptibility rates to gentamycin, erythromycin, clindamycin, tetracyclines, rifampicin and quinolones were ranged from 15. 8% to 59. 6%. All Staphylococci isolates were susceptible to teicoplanin, vancomycin, linezolid, daptomycin and tigecycline. All Enterococcus isolates were susceptible to daptomycin and tigecycline. All E. faecalis ( 158/158 ) and 96. 4% ( 133/138 ) of E. faecium were susceptible to teicoplanin. About 98. 0% ( 150/153 ) of E. faecalis and 97. 1% ( 145/138 ) of E. faecium were susceptible to linezoild. About 45. 8% (70/153) of E. faecalis and 60. 9% (84/138) of E. faecium were resistant to gentamycin with a high concentration. The susceptibility of E. faecalis to all the antibiotics tested exceptchloramphenicolandtetracyclinewashigherthanthatofE.faecium.Conclusions Basedon different age groups and regions, the resistance rates of Gram-positive cocci are different. Teicoplanin, vancomycin, tigecycline, daptomycin, linezolid and tedizolid showed very high activity against Gram-positive cocci. (Chin J Lab Med,2015,38:373-381)

Objective To investigate antimicrobial resistance among gram-positive cocci in 14 teaching hospitals in China in 2011.Methods From June 2011 to December 2011,1498 consecutive and non-repetitive gram-positive cocci were collected from 14 teaching hospitals.The minimal inhibitory concentration (MIC) of antibacterial agents was determined by agar dilution method.A retrospective study was conducted on rates of resistance to antimicrobial agents.Data was compared using chi-square test.Results The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) and methicillinresistant coagulase-negative Staphylococci (MRCoNS) was 43.7％ (222/508),and 85.6％ (214/250),respectively.The MRSA prevalence ranged from 20.0％ to 63.5％ in different regions.About 58.2％ (82/141) of Staphylococcus aureus from respiratory tract specimens,44.8％ (48/107) of Staphylococcus aureus from blood samples,and 23.8％ (31/130) of Staphylococcus aureus from pus and wound were resistant to methicillin.The susceptible rates of MRSA to chloramphenicol and sulfamethoxazole-trimethoprim SXT were 94.1％ (209/222) and 83.3％ (185/222),respectively.Susceptibility to gentamycin,erythromycin,clindamycin,tetracyclines,rifampicin and quinolones were from 11.3％ to 52.3％.All Staphylococci isolates were susceptible to vancomycin,teicoplanin,linezolid and daptomycin.Five vancomycin-resistant enterococcus (VRE) strains were found in this study.All enterococcus isolates were susceptible to daptomycin(268/268),and 98.3％ (118/120) of E.faecalis and 99.3％ (147/148) of E.faecium were susceptible to linezoild.About 45.9％ (68/148) of E.faecalis and 67.5％ (81/120) of E.faecium were resistant to high concentration gentamycin.The susceptibility of E.faecalis to all the antibiotics except for chloramphenicol and tetracycline was higher than that of E.faecium.The prevalence of penicillinnonsusceptible Streptococcus pneumoniae (PNSSP) was 15.5％ (37/239).The prevalence of PNSSP in children below 3 years-old was 25％ (13/52),and the prevalence of PNSSP from other patients was 13％(24/187).About 91.6％ (219/239),88.7％ (212/239) and 88.3％ (211/239) of S.pneumonia was resistant to erythromycin,clindamycin and tetracyclines.All S.pneumoniae strains were susceptible to teicoplanin,vancomycin,linezolid,tigecycline and daptomycin.Penicillin still showed high activity against Streptococcus spp.β-hemolytic group.More than 60％ of Streptococcus.spp.β-hemolytic group are resistant to erythromycin,clindamycin and tetracyclines.Conclusions Based on regions,the resistance rates of Gram-positive cocci are different,of which,the increasing tendency should be taken seriously.Teicoplanin,vancomycin,linezolid,tigecycline and daptomycin show very high activity against Gram-positive cocci.