Identification of in vivo-induced antigen Tp0462 of Treponema pallidum and evaluation of its application to serodiagnosis of syphilis / 中华皮肤科杂志

Objective To screen and identify the in vivo-induced antigen Tp0462 of Treponema pallidum (Tp),and to evaluate its value for clinical serological diagnosis of syphilis.Methods Genomewide DNA was extracted from the Tp Nichols strain,and polymerase chain reaction (PCR) was performed to amplify the Tp0462 gene.A recombinant plasmid pET30a (+)-Tp0462 was constructed and transfected into the Escherichia coli (E.coli) Rosetta (DE3) strain.Recombinant protein Tp0462 was abundantly expressed,purified and identified.A total of 18 New Zealand rabbits were randomly and equally divided into 3 groups:viable Tp-incubating group incubated with viable Tp in the testes,inactived Tp-incubating group incubated with ultraviolet irradiation-killed Tp in the testes,and control group receiving no treatment.After incubation,blood samples were collected at different time points,and the sera were isolated for identification of characteristics of in vivo-induced antigen Tp0462.Enzyme-linked immunosorbent assay for Tp0462 (Tp0462-ELISA),Treponema pallidum particle agglutination assay (TPPA),preliminary syphilis screening ELISA and rapid plasma reagin (RPR) card test were applied in 336 clinical serum samples from patients with syphilis,so as to preliminarily evaluate the value of Tp0462 for the diagnosis of syphilis.Results The optimum conditions for expression of the recombinant plasmid Tp0462-pET30a(+) in E.coli Rosetta (DE3) strains were the treatment with 0.5 mmol/L isopropyl thiogalactoside (IPTG) on a shaker at 180 rpm for 4 hours.In the viable Tp-incubating group,the serum level of specific anti-Tp0462 antibody sharply increased from week 2,and went steady after week 5.However,the specific anti-Tp0462 antibody maintained a low level in the inactived Tp-incubating group and the control group.The viable Tp-incubating group showed a significantly higher level of specific anti-Tp0462 antibody compared with the inactived Tp-incubating group and the control group (both P ＜ 0.05),while no significant difference was observed between the inactived Tp-incubating group and the control group (P =0.256).The level of anti-Tp92 antibody was significantly higher in the viable Tp-incubating group and the inactived Tp-incubating group than in the control group (P ＜ 0.05),while there was no significant difference between the viable Tp-incubating group and the inactived Tp-incubating group (P =0.127).Compared with TPPA,the sensitivity,specificity,consistency rate and area under the curve (AUC) of Tp0462-ELISA for the diagnosis of syphilis were 91.7％,98.8％,95.2％ and 0.997 respectively.Tp0462-ELISA was consistent to preliminary syphilis screening ELISA and RPR with a Kappa coefficient of 0.846 and 0.293,respectively.Conclusion Tp0462-ELISA has shown evidently higher sensitivity and specificity in the serodiagnosis of syphilis,and Tp0462 can serve as promising antigens for the diagnosis of syphilis.

Comparison of RPR′s performance in syphilis screening between pregnant women and non-pregnant women / 国际检验医学杂志

Objective To study comparatively the performance of rapid plasma reagin (RPR) test in syphilis detection among pregnant women and non-pregnant women to provide reference for detecting syphilis in pregnant women.Methods The women aged 20-40 years old were selected and divided into the pregnant group and the non-pregnant group.RPR and treponema pallidum particle assay(TPPA) were simultaneously adopted to conduct the syphilis detection.The positive cases were judged by the TPPA detection results combined with the contact history,clinical symptoms and treatment situation.The results were compared with those by RPR for determining the false negative and false positive in RPR.The false negative rate and false positive rate of RPR detection results were analyzed in the two groups.Results Among 117 pregnant women,15 cases were false negative in RPR and 9 cases were false positive in RPR;among 755 non-pregnant women,there were 44 cases of false negative RPR results and 8 cases of false positive RPR results.The false negative rates in the pregnant group and non-pregnant group were 25.0% and 8.8% respectively,the difference was statistically significant(χ2=14.739,P<0.05);the false positive rates in the pregnant group and non-pregnant group were 15.7% and 3.1% respectively,the difference was statistically significant (χ2=14.722,P<0.05).Conclusion There are many factors affecting RPR for detection syphilis,pregnant women are the specific group,so higher false positive rate and false negative rate exist than non-pregnant women,the detection results should be comprehensively judged by combining with clinical symptoms and disease history,if necessary,combining with other syphilis detection method for avoiding missed diagnosis and misdiagnosis.

Clinical distribution and antimicrobial resistant characteristics of Pseudomonas aeruginosa in Northern Guangdong / 国际检验医学杂志

Objective To investigate the clinical distribution and antimicrobial resistant characteristics of Pseudomonas aeruginosa(PA)in the northern area of Guangdong.Provide the reference for clinical to prevent infection and reasonable choice of antibiotics and reduce the production of drug resistance strains.Methods The separation and identification of PA were performed by conventional methods during the data of drug 2013 and 2014.The data of sensitivity test of PA were analyzed by WHONET 5.6 and SPSS19.0 softwares.Results The 584 strains PA were mainly distributed in ICU,department of orthopaedics and respiratory medicine.Specimens were mainly from sputum and wound secretion.The detection of PA to 12 antibacterial agents showed different resistance.The antimicrobial with highest resistance was the gentamicin and lower resistance rates to fluoroquinolones,carbapenems,enzyme inhibitors.And a downward trend was shown in drug resistance to CIP,FEP,LEV,SCF.Conclusion PA mainly cause lung and wound infection,especially those old patients that come from ICU,department of orthopaedics and respiratory medicine.Although the drug resistance rates of PA to the commonly used antibiotics are relatively low,The clinicians should reasonably use antibiotics so as to reduce the resistant strains,especially the produce of MDR-PA and PDR-PA.

Drug Resistance Spectrum Analysis of 2 527 Clinical Isolates of Bacteria / 现代检验医学杂志

Objective To investigate the resistance of clinical isolates from Yuebei People’s Hospital so as to provide the ref-erence for clinical use of antibiotics.Methods To comprehensively analyze the drug resistance,AST was performed with K-B or MIC method to 2 527 strains of clinical isolates and the data were analyzed by WHONET 5.4 and SPSS19.0 softwares according to the breakpoints of CLSI 2012 standard.Results MRSA accounted for 30.9% in Staphylococcus aureus iso-lates,MRCNS accounted for 76.2% incoagulase negative Staphylococcus isolates ,no detection of vancomycin or linezolid re-sistant strains.The antimicrobial drug resistance of Enterococcus faecium was significantly higher than that of Enterococcus faecalis ,no detection of glycopeptide and linezolid resistant strains in Enterococcus faecalis ,but one linezolid resistant strains and two teicoplanin resistant strains in Enterococcus faecalis .Enterobacteriaceae remain highly sensitive to carbapen-em antibiotics,ESBLs produced strains in Escherichia coli ,Klebsiella pneumoniae were 43.9% and 37.5% respectively,five polymyxin B resistant strains were detected in Pseudomonas aeruginosa ,and 63.7% of MDRO rate in Acinetobacter bau-mannii .Conclusion Drug resistance of clinical pathogenic bacteria remain seriously in hospital,clinical application of antibi-otics should be reasonable based on the result of drug sensitivity.

Expression, purification and identification of recombinant Omp~2 of Chlamydia trachomatis / 中华检验医学杂志

Objective To express outer membrane protein 2(Omp2) of Chlamydia trachomatis, purify expressed products and study its immunity.Methods The target gene encoding Omp2 167—434 amino acid residues was amplified by PCR from C. trachomatis template DNA. The targeted DNA fragment was cloned into expression vector pET28b(+) and introduced into competent E. coli BL21(DE3) cell. Recombinant Omp2aa_ 167 ～aa_ 434 was expressed after induction by IPTG and analyzed by SDS-PAGE and Western blot, purified with Ni-NTA-His affinity chromatography. The rOmp2aa_ 167 ～aa_ 434 was used to immune rabbits for immunogenicity assessment.Results Restriction enzymes cleavage analysis and DNA sequencing confirmed that the plasmid pET28b(+)/Omp2aa_ 167 ～aa_ 434 was correctly constructed. The 35.0?103 molecular weight pure protein, which specifically reacted with serum from C. trachomatis infected patient by Western blot, was obtained by optimizing the conditions for both expression and purification. The titer of serum antibodies was above 1∶1 280 as detected by ELISA.Conclusion The expressed product showed good immunity.

Study of isolation of fluoroquinolone-resistant Ureaplasma urealyticum and identification of mutant sites / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To study the resistance mechanism of clinical isolates of Ureaplasma urealyticum resistant to fluoroquinolones.</p><p><b>METHODS</b>Thirteen isolates of Ureaplasma urealyticum resistant to six fluoroquinolones were selected out of 184 clinical isolates and their QRDRs (quinolone resistance-determining region) gyrA, gyrB, parC and parE were amplified by PCR. Sequencing results were compared to those susceptible reference strains and a comparison of deduced amino acid sequences were performed.</p><p><b>RESULTS</b>Sequence comparison revealed a C to A change at 87nt of gyrA QRDR leading to the substitution of Asp95 with glutamic acid and a C to T change at 50nt of parC QRDR leading to the substitution of Ser80 with leucine.</p><p><b>CONCLUSION</b>These results suggest that a C to A change at 87nt of gyrA QRDR and a C to T change at 50nt of parC QRDR are associated with fluoroquinolone resistance of Ureaplasma urealyticum.</p>

Topoisomerase gene mutation and mechanism of ureaplasma urealyticum resistant to quinolones / 中华检验医学杂志

Objective To study the relation of type Ⅱ topoisomerase gene mutation and mechanism of Ureaplasma urealyticum resistant to quinolones. Methods 13 isolates of Uu resistant to 6 quinolones were selected from 184 clinical isolates by using broth dilution method, and their gyrA, gyrB, parC, parE were amplified by PCR.After sequencing, results were compared with the nucleotide sequence of susceptible reference strain. ResultsMICs of resistant Uu isolates were 4 to 32 fold higher than susceptible reference strain counter parts; sequence comparison revealed a C to A change at 87nt of gyrA QRDR led to the substitution of aspartic acid by glutamic acid and a C to T change at 50nt of parC QRDR led to the substitution of serine by leucine, with no amino acid change observed in GyrB and ParE. Conclusion These results suggest that a C to A change at 87nt of gyrA QRDR and a C to T change at 50nt of parC QRDR are associated with quinolone resistance of Uu.