Objective To detect point mutations of TAC1 gene in fluconazole-resistant Candida albi-cans isolates with rolling circle amplification (RCA), develop an accurate, rapid and specific assay to detect single nucleotide polymorphisms (SNPs), and to estimate the relationship between mutations of TAC1 gene and resistance to fluconazole. Methods A total of 33 fluconazole-resistant Candida albicana isolates, including 8 strains from America and 25 from Australia, were collected. Four TAC1-specific padlock probes were designed according to previously reported mutations. DNA was extracted from these tested strains, subjected to amplification of three targeted fragments of TAC1 gene with PCR. Then, RCA was performed to detect point mutations of TAC1 gene in resistant Candida albicans strains. At the same time, the target fragments underwent sequencing analysis, and the results of RCA were compared with those of sequencing. Results Two types of resistance-associated mutations were found in 5 out of the 33 fluconazole-resistant strains. Among the 5 strains, 4 were from America, 1 harbored T225A mutation and 4 carried A736V mutation. No related mutation was found in TAC1 gene of 4 fluconazole-sensitive isolates. Conclusions RCA assay could accurately and rapidly detect point mutations of genes. Further studies are required to clarify the relationship between TAC1 point mutations and fluconazole resistance.

Objective To detect antibodies against chlamydial plasmid-encoded protein 3 (Pgp3),outer membrane complex protein B C-terminal peptide (OmcBc),CT841 protein and heat shock protein 60 (HSP60) in the sera of patients with urogenital Chlamydia trachomatis infection.Methods Recombinant plasmids encoding the aforementioned four proteins and an empty plasmid were transformed into Escherichia coli separately followed by 2-hour isopropyl-1-thio-β-galactopyranoside (IPTG) induction and cell lysis.The expressed proteins were purified with glutathione magnetic beads and then used to coat 96-well enzyme-linked immunosorbent assay (ELISA) plates precoated by glutathione.Serum samples were collected from 20 patients with and 20 clients without urogenital C.trachomatis infection attending the sexually transmitted disease (STD) clinic of Tianjin Medical University General Hospital.ELISA with the expressed protein-coated plates was adopted to detect antibodies against these proteins in the serum samples.Results Of the 20 serum samples from C.trachomatis-infected patients,14 (70％)had anti-Pgp3 antibody,9 (45％) anti-OmcBc antibody,8 (40％) anti-CT841 antibody,and 5 (25％) anti-HSP60 antibody.Meanwhile,no antibody was detected in any of the serum samples from uninfected clients except for one with anti-HSP60 antibody.Conclusions Of the four studied C.trachomatis proteins,Pgp3 appears to have the strongest antigenicity with the highest antibody-detection rate,while HSP60 exhibits the weakest antigenicity with the lowest antibody-detection rate.

ObjectiveTo study the effect of a recombinant plasmid encoding mouse interleukin 2 (mlL-2) on the immunogenicity of DNA vaccine against Chlamydia trachomatis(Ct) serovar E.Methods BALB/c mice were divided into 4 groups to be intramuscularly inoculated with blank plasmid(negative control group),DNA vaccine against Ct serovar E(DNA vaccine group),DNA vaccine against Ct serovar E and a recombinant plasmid containing mIL-2(combination group),and inactivated Ct serovar E elementary bodies (positive control group),respectively.The immunological effects were evaluated by posterior foot pad thickness,proliferation level of spleen lymphocytes,serum level of IL-4 and interferon (IFN)-γ in mice,and the capability to clear Ct genital tract infection.ResultsThe proliferation index of spleen lymphocytes in the combination group and positive control group was similar(3.64 ± 0.41 vs.3.77 ± 0.34),but was significantly different from that in the blank control group and DNA vaccine group (1.37 ± 0.21 and 2.52 ± 0.30).The serum level of IL-4 was(38.49 ± 12.24) pg/ml in the positive control group,significantly higher than in the negative control group,DNA vaccine group and combination group ((25.37 ± 18.93),(24.75 ± 8.49),(21.74 ± 6.43) pg/ml,respectively).With respect to the serum level of IFN-γ,the combination group and positive control group were similar ((1923.3 ± 518.1) pg/ml vs.(2712.5 ± 887.2) pg/ml),but were significantly different from the negative control group and vaccine group((310.8 ± 160.7) pg/ml and(601.3 ± 357.9) pg/ml).Six days after Ct challenge,the exfoliated cells from genital tract were positive for Ct culture in the negative control group,but negative in the other 3 groups.ConclusionIL-2 genetic adjuvant can enhance the immune response,especially Th1 type response,induced by the DNA vaccine against Ct serovar E.

ObjectiveTo analyze the association of HLA-DQA1 gene polymorphism with persistent Chlamydia trachomatis genital infection.Methods Blood samples were collected from 80 patients with persistent genital Chlamydial infection,80 patients with common genital Chlamydial infection(who tested negative for Chlamydia trachomatis after one course of standard systemic treatment) and 80 normal human controls.HLA-DQA1 alleles were genotyped by PCR followed by gene sequencing.ResultsThe frequency of HLA-DQA1*0102 allele and HLA-DQA1*0501 allele was 22.5％ and 5.0％ respectively in patients with persistent genital Chlamydial infection,5％ and 20％ respectively in those with common genital Chlamydial infection,2.5％ and 17.5％ respectively in normal human controls.Compared with the patients with common genital Chlamydial infection and controls,the patients with persistent genital Chlamydial infection had a higher frequency of HLA-DQA1*0102(x2 =14.6286,P ＜ 0.001 ),but a lower frequency of DQA1*0501 (x2 =6.2598,P ＜ 0.05).ConclusionsHLA-DQA1*0102 allele may be a susceptible gene or closely linked with the susceptible genes of persistent genital Chlamydial infection.HLA-DQA1*0501 allele may have protective effects against persistent genital Chlamydial infection.

Objective To study cellular immune responses induced by DNA vaccine against Chlamydia trachomatis (Ct) serotype E. Methods BALB/c mice were divided into three groups to be intramuscularly immunized by blank plasmid (negative control group), DNA vaccine against Ct serotype E (vaccine group), and inactivated Ct elementary body (positive control group), respectively. Two weeks after the last immunization,delayed-type hypersensitivity (DTH) response was evaluated; MTT assay was performed to detect the proliferation of spleen lymphocytes, ELISA to measure the serum level of interferon-γin mice. Some immunized mice underwent a genital challenge with Ct elementary body followed by isolation of Ct from exfoliated epithelial cells in genital tract and pathological examination of cervical tissue from the challenged mice. Results Compared to negative control group, vaccine group and positive control group experienced a stronger DTH response.The lymphocyte stimulating index and serum level of IFN-γwere highest in the positive control group (3.81 ±0.30, 2891.7 ± 1048.8 μg/L), followed by vaccine group (2.35 ± 0.25, 593.3 ± 342.6 μg/L) and negative control group (1.48 ± 0.15, 309.2 ± 157.9 μg/L), and significant difference was observed between the three groups (P ＜ 0.05 or 0.01 ). After Ct challenge, Ct was isolated from exfoliated epithelial cells and cervical tissue was damaged in the negative control group, while in the other two groups, Ct was undetected and genital tract tissue was intact. Conclusions The DNA vaccine against Ct serotype E could induce Ct-specific cellular immune responses to some extent, and offer a protection against vaginal challenge with Ct.

ObjectiveTo compare the recovery rates calculated according to different criteriain patients with urogenital Chlamydia trachomatis (Ct) infection after treatment with azithromycin. Methods Clinical data on outpatients who were diagnosed with urogenital Ct infection and treated with azithromycin in the sexually transmitted disease(STD) outpatient clinic of Tianjin Medical University General Hospital were retrospectively reviewed. Recovery rates were calculated according to the improvement of symptom and (or) reexamination results of Ct at 1,5 and 9 weeks after the end of treatment.ResultsSignificant differences were observed between the recovery rates calculated according to symptom improvement and those according to laboratory reexamination results.No obvious correlation existed between the presence of symptom and positive reexamination results.The recovery rates calculated according to the second reexamination result differed significantly from those according to the first reexamination result,but were similar to those according to the third reexamination result. ConclusionsThe cure of Ct infection should be determined according to laboratory test results,and two times of reexamination at 1 and 5 weeks after the final treatment are recommended.

Objective To explore diagnostic value of the serum and urine procalcitonin (PCT) detecting in the urinary system infection.Methods The serum and urine PCT levels in 45 urinary system infection patients with clear pathological diagnosis (exclude other system infections) who were outpatiented or hospitalized in the People's Hospital of Zhongshan between March and November 2016 (including 21 cases of upper urinary tract infection and 24 cases of lower urinary tract infection) and 35 healthy adults who went through physical examinations at the hospital during the same period,were measured using electrochemiluminescence immunoassay (ECLIA) on Cobase 601 Immunoassay Analyzer and analyzed to compare the differences of PCT levels in the three groups.Results The urine PCT level in upper urinary tract infection group was 0.243± 0.123 ng/ml.It was significantly lower than lower urinary tract infection group (0.486±0.232 ng/ml,t=4.11,P=0.000) and control group (0.454± 0.253 ng/ml,t=3.96,P=0.000).The serum PCT level in upper urinary tract infection group was 0.062±0.014 ng/ml.It was obviously higher than that in lower urinary tract infection group (0.043±0.020 ng/ml,t=3.56,P=0.01) and control group (0.032±0.013 ng/ml,t=7.38,P=0.000).In all groups,the urine PCT levels were significantly higher than their serum PCT levels (t =9.48,9.12,6.79,P＜ 0.01),and significant differences were observed in them.The sensitivity,specificity,positive predictive value and negative predictive value of serum PCT for diagnosing upper urinary tract infections were 81.5％,84.2％,80.6％ and 85.6％ respectively,and the urine PCT were 86.4％,80.7％,88.4 ％ and 83.1 ％ respectively.Conclusion Detection of serum and urine PCT has important accessory diagnostic value for identifying upper and lower urinary tract infections.

Objective To detect the antibodies against recombinant chlamydial plasmid-encoded protein 3(rPgp3),chlamydial protease-like activity factor(rCPAF),Ct143 encoded protein(rCT143), Ct101 encoded protein(rCT101),Ct694 encoded protein(rCT694),Ct813 encoded protein(rCT813), Chlamydia membrane protein A(rIncA),Ct875 encoded protein(rCT875),major outer membrane protein (rMOMP)and heat shock protein 60( rHsp60)in serum samples collected form patients with urogenital Chlamydia trachomatis(Ct)infection and to evaluate the antigenicity of those proteins. Methods The re-combinant plasmids expressing the 10 proteins and a blank plasmid were transformed into E. coli BL21 strains,respectively. The transformed E. coli BL21 strains were induced by isopropyl β-D-1-thiogalactopyr-anoside(IPTG)to express recombinant proteins. The glutathione pre-coated 96-well ELISA plates were coa-ted with lysates. Serum samples were collected from 50 patients with Ct infection and 10 patients without Ct infection. ELISA was performed to detect the antibodies against 10 recombinant proteins. Results The anti-bodies against rPgp3,rCPAF,rCT143,rCT101,rCT694,rCT875,rCT813,rMOMP,rIncA and rHsp60 proteins were respectively detected in 44 cases(88% ),38 cases(76% ),37 cases(74% ),36 cases (72% ),33 cases(66% ),31 cases(62% ),30 cases(60% ),26 cases(52% ),24 cases(48% )and 17 cases(34% )out of 50 serum samples. No antibodies against 10 recombinant proteins were detected in the serum samples collected from patients without Ct infection. Conclusion The rPgp3 protein showed the strongest antigenicity among all of the studied proteins,followed by rCPAF and rCT143 proteins. The rHsp60 protein showed the lowest antigenicity.

Objective: To summarize the experience of valve replacement for multiple valve insufficiency in patients with giant left ventricle and the operative indication. Methods: Multiple valvular operations were performed in 62 patients with giant left ventricle between 1991 and 2002. Combined mitral and aortic valve replacement was performed in 56, tricuspid valve annuloplasty in 43, left atrium placation surgery in 12, and mitral valve replacement in 5. Results: The early postoperative complication and mortality rate were 45.2% and 17.7%, respectively .The late mortality rate was 6.5%. The main factors influencing the early surgical results were preoperative severe left ventricular enlargement (ESD ≥6.0cmand EDD ≥8.0cm) and systolic dysfunction (EF ≤0 40 and FS ≤0 25), perioperative ventricular fibrillation, postoperative low cardiac output and multiple organal failure. The main factors affecting long term survival were postoperative severe ventricular arrhythmia and left ventricular enlargement with depressed systolic performance. Conclusion: The keys to improve the early and late results of multiple valve replacement in these patients with giant left ventricle are the choice of optimum surgical timing, the proper management of the high risk factors mentioned above during perioperative and follow up periods.

Objective To investigate the feasibility of C.trachomatis culture in HaCaT human keratinocytes.Methods According to the procedure for C.trachomatis culture in McCoy cells,clinical swab specimens and standard strains of C.trachomatis serotype E were inoculated into HaCaT cells.Iodine staining,a fluorescent monoclonal antibody test and PCR amplification of the endogenous plasmid of C.trachomatis were performed to detect the growth of C.trachomatis in HaCaT cells.Five passages of subculture were carried out for the standard strain of C.trachomatis serotype E in HaCaT cells,and inclusion bodies were counted after each passage.One-factor analysis of variance was conducted by using the software SPSS17 to determine if C.trachomatis was propagated in HaCaT cells.Results Iodine staining showed typical inclusion bodies of C.trachomatis in the cytoplasm of HaCaT cells.Yellow fluorescence-labeled granules were observed in the HaCaT cells under a microscope.Endogenous plasmids of C.rachomatis were successfully amplified by PCR from the infected HaCaT cells.The number of inclusions in HaCaT cells gradually increased at passage 1 through 5.Conclusions C.trachomatis is successfully cultivated in HaCaT cells in vitro,and the standard strain of C.trachomatis serotype E can propagate in HaCaT cells.