Overview of resistance mechanisms in Campylobacter / 中华预防医学杂志

Campylobacter is a major cause of food-borne gastroenteritis worldwide. While mortality is low when people was infected with Campylobacter, morbidity imparted by post-infectious sequelae such as Guillain-Barré syndrome and irritable bowel syndrome is significantly noteworthy. Although fluoroquinolones and macrolides were the first line drug for the treatment of Campylobacter infections, there is a tough challenge in clinical treatment with high antimicrobial resistant rate and multi antimicrobial resistance arise. Based on the latest literature acquired in this work, we have chosen five classes of antibiotics always used in clinical, and discussed antibiotic resistance mechanisms and transmission of Campylobacter, in order to provide proper therapy both in the veterinary and human populations, and support basis data for the development of new drugs.

Genotyping of Chlamydia trachomatis strains from male patients with urethritis in sexually transmitted disease clinic / 中华皮肤科杂志

Objective To investigate the serovar distribution of Chlamydia trachomatis (Ct) isolated from male patients with urethritis in sexually transmitted disease (STD) clinic.Methods Urine specimens were collected from male patients with urethritis in STD clinic at Hospital of Dermatology,Chinese Academy of Medical Sciences between January 2013 and December 2013.Fluorescence-based quantitative PCR was performed to detect Ct DNA in these specimens.DNA was extracted from Ct-positive urine specimens,and nested PCR was conducted to amplify the VS1-VS2 regions of the outer membrane protein A (ompA) gene,followed by gene sequencing.The resulting sequences were aligned to reference sequences by the DNAStar5.0 software to determine Ct serovars.Results A total of 432 urine specimens were collected,and 33.1％ (143/432) of them were positive for Ct.The VS1-VS2 regions of the ompA gene were amplified from 127 out of the 143 Ct-positive specimens,but not from the other 16 specimens.Nine serovars were identified by gene sequencing among the 127 specimens,including serovar E (29 strains,22.83％),F (28 strains,22.05％),D (19 strains,14.96％),G (16 strains,12.60％),J (16 strains,12.60％),K (8 strains,6.30％),H (5 strains,3.94％),I (3 strains,2.36％) and B (3 strains,2.36％),and Ct serovars E,F,D,J and G accounted for 85.02％ among all the strains.Synonymous mutations were identified in 14 out of the 127 strains when compared with reference strains.Conclusions E,F,D and G serovars were the main Ct serovars in male patients with urethritis in STD clinic.The proportion of Ct serovar E strain was decreased,but that of serovar J strain was increased compared with 20 years ago.

Detección de Chlamydia trachomatis en muestras de exudado endocervical mediante una prueba de diagnóstico rápido y dos técnicas de reacción en cadena de la polimerasa / Detection of Chlamydia trachomatis in endocervical swab using rapid test and two reaction techniques polymerase chain

Introducción: Chlamydia trachomatis es el principal agente bacteriano que produce infecciones de transmisión sexual.Objetivo: detectar la presencia de C. trachomatis utilizando una prueba de diagnóstico rápido y compararla con la reacción en cadena de la polimerasa (RCP).Métodos: se procesaron 50 muestras de exudado endocervical, de mujeres sintomáticas del municipio 10 de Octubre. A las muestras se les aplicó la prueba Chlamy-check-1, un ensayo de RCP del gen del plásmido críptico y una RCP en tiempo real (RCP-TR) de la proteína mayor de la membrana externa (MOMP) de C. trachomatis, que fue utilizada como referencia. Se calculó, sensibilidad, especificidad, valor predictivo positivo (VPP) y negativo (VPN).Resultados: de las muestras estudiadas, 44 resultaron positivas por la prueba rápida, mientras que por la RCP del plásmido críptico solo 3 muestras (6 porciento) amplificaron. Al aplicar la RCP-TR, 4 muestras (8 porciento) se confirmaron como positivas, coincidiendo 3 por los tres métodos de diagnóstico. Al evaluar la prueba Chlamy-check-1 frente a la prueba de referencia se observó una sensibilidad de 100 porciento, mientras que la especificidad fue de 13 porciento, así como un VPP de 9,1 porciento y VPN de 100 porciento. Por el contrario, la RCP del plásmido críptico mostró una sensibilidad y especificidad de 75 y 100 porciento, respectivamente; un VPP de 100 porciento y VPN de 97,9 porciento.Conclusiones: se obtuvo diferencia entre los porcentajes de positividad detectados con la prueba rápida, y las técnicas de RCP. La baja especificidad de la prueba rápida indica la necesidad de realizar estudios de evaluación de este estuche diagnóstico.

Introduction: Chlamydia trachomatis is the leading bacterial agent that causes sexually transmitted infections.Objective: to detect the presence of C. trachomatis using a rapid test and compare it with the chain reaction (PCR).Methods: 50 endocervical exudates taken from symptomatic women were processed in Diez de October municipality. The samples were applied the Chlamy-check-1 test, a PCR assay of the cryptic plasmid gene and a real-time PCR (RT-PCR) of major outer membrane protein (MOMP) of C. trachomatis which was used as reference. Sensitivity, specificity, positive (PPV) and negative (NPV) predictive value were calculated.Results: 44 samples were positive by the rapid test, whereas only three samples (6 percent) amplified by cryptic plasmid PCR. Applying RT-PCR, 4 samples (8 percent) were confirmed as positive, 3 samples matched with three diagnostic methods. In assessing the Chlamy-check-1 versus the reference test, 100 percent of sensitivity was observed, while the specificity was 13 percent> Also PPV was 9.1percent and NPV was 100 percent. On the contrary, the cryptic plasmid PCR had 75 and 100 percent of sensitivity and specificity respectively, 100 percent PPV and 97.9 percent NPV.Conclusions: the difference was obtained between the percentages of positivity detected with both the rapid test, and CPR techniques. The low specificity of the rapid test indicates the need for further studies to evaluate this diagnostic kit.

Immune responses induced by DNA vaccine combined with recombinant adenovirus containing MOMP gene of Chlamydia trachomatis serovar E / 中华微生物学和免疫学杂志

Objective To investigate the specific immune responses induced by DNA vaccine com-bined with recombinant adenovirus carrying major outer membrane protein ( MOMP) gene of Chlamydia trachom-atis (Ct) serovar E.Methods Recombinant eukaryotic expression plasmid pVAX 1-MOMP and recombinant adenovirus Ad-MOMP were constructed and purified .Four immunization strategies were designed including DNA immunization (DNA group), recombinant adenovirus immunization (Ad group), DNA prime-recombinant ade-novirus boost regimen ( DNA/Ad group ) and recombinant adenovirus prime-DNA boost regimen ( Ad/DNA group) .Two weeks after the final immunization , vaginal wash specimens , blood samples and spleens were col-lected from all mice for the evaluation of humoral and cellular immune responses .Results Th1 responses and mucosal responses were not found in DNA group .Ad group induced both Th 1 responses and local mucosal re-sponses , and its IgG and IgA levels were significantly higher than those in DNA group .DNA/Ad group and Ad/DNA group induced stronger Th 1 responses and humoral responses than DNA and Ad group .Serum antibodies and mucosal antibodies in DNA/Ad group were higher than those in Ad/DNA group, but Th1 responses in DNA/Ad group were impaired than those in Ad/DNA group.Conclusion The recombinant adenovirus could induce Th1 responses and genital mucosal responses .Combined immunization of DNA/Ad or Ad/DNA showed enhanced specific immune responses in comparison with the single immunization with DNA or Ad .The differ-ences between the two combination methods were that DNA /Ad group stimulated higher levels of specific anti-body responses , while Ad/DNA group produced stronger Th 1 responses .The study provided a reference for the enhancement and development of vaccines against Chlamydia trachomatis in further studies .

The cross immune responses of the rhesus monkey induced by the E style Chlamydia trachomatis recombinant major outer membrane protein / 中华微生物学和免疫学杂志

Objective To test cross immune responses induced in rhesus monkeys immunized with the recombinant major outer membrane protein(rMOMP).Methods Six rhesus monkeys were divided into three groups:the group vaccinated with purified rMOMP and Freund's adjutants,the group vaccinated with Freund's adjutants only and the control group vaccinated with PBS.All of the rhesus monkeys vaccinated intramuscularly at 0,2,4 weeks.Two weeks after the last time,The IFN-γand Chlamydia-specific antibody titers in sera,which were determined by ELISA,lymphocyte proliferation assay were performed by MTT,and observ the delayed hypersensitivity and in vitro neutralization assays.Results The result of the monkeys immunized with rMOMP and Freund's adjuvant:the specific immune responses can be observed.The in vitro neutralization and lymphocyte proliferation assays were observed better in the same group.Conclusion After being vaccinated with rMOMP,the monkeys can develop strong and effective Chlamydia-specific cross immune responses.

The cellular immune response produced in BALB/c mice immunized with HPV6b L1/Ct MOMP multi-epitope chimeric DNA / 中华微生物学和免疫学杂志

Objective To study on the specific cellular immune response produced in BALB/c mice immunized with human papillomavirus (HPV) type 6b capsid protein L1 and Chlamydia trachomatis (Ct) major outer membrane protein(MOMP) multi-epitope chimeric DNA (HPV6b L1/Ct MOMP multiepitope) , and the enhancement of the specific cellular immune response to Ct MOMP multi-epitope by HPV6b L1. Methods The Ct MOMP multi-epitope gene was connected to the C terminal of HPV6b L1,the gene of HPV6b L1 had been optimized according to the codon usage of eukaryotic system, and then the HPV6b L1/Ct MOMP multi-epitope chimeric gene was cloned to pcDNA3.1 ( + ) vector. After identification by restriction enzyme digestion and DNA sequencing, the recombinant plasmid was transfected into COS-7 cells, Indirect Immunofluorescence (IIF) was used to confirm the expression of proteins. Then, BALB/c mice were randomly assigned to receive (intramuscular injection) either pcDNA3.1 ( + )/HPV6b L1/Ct MOMP or pcDNA3.1 ( + )/Ct MOMP or pcDNA3.1 ( + ) or PBS ( n = 12, 150 μg/time), and the same immunization schedule was repeated third times at 2 week intervals. The level of cytokine( IFN-γ, IL-4, IL-10) -producing CD3+ T cells in spleen, the cytotoxicity of Ct MOMP-specific and HPV6b L1-specific cytotoxic T lymphocyte (CTL) in spleen were detected by intracellular cytokine staining-fluorescence activated cell sorter (ICS-FACS) and LDH release assays, respectively. Results After immunization, when the efCTL (44.56%±4.02%, 35.35% ±2.89% ) and HPV6b L1 specific cytotoxicity of CTL (27.08% ±2.04%, 21.68% ±4.06% ) in pcDNA3.1 ( + )/HPV6b L1/Ct MOMP multi-epitope chimeric DNA immunized mice, were significantly higher than that in pcDNA3.1 ( + )/Ct MOMP multi-epitope DNA (35.50%±2.68%, 30.24% ±1.75%; 12.27% ±3.36%, 9.32% ±3.07%) and other control groups(F=72.87, F=114.55, P＜0.05; F=30.04, F=10.47, P＜0.05), and Ct MOMP multi-epitope specific cytotoxicity of CTL in pcDNA3.1 ( + )/Ct MOMP multi-epitope DNA immunized mice were significantly higher than that in control groups( F = 58.85, F = 120.21; P<0.05). The level of intracellular cytokine IFN-γ in pcDNA3.1 ( + )/HPV6b L1/Ct MOMP multi-epitope DNA immunized mice(4.34% ±0.06%)was higher significantly than that in pcDNA3.1 ( + )/Ct MOMP multi-epitope DNA immunized mice(3.14% ± 0.18%, P<0.05 ) and other control groups ( F = 473.83, P<0.05 ), while, the levels of IL-4 ( F =0.97, P ＞ 0.05 ) and IL-10 ( F = 2.25, P ＞ 0.05 ) had no significant difference between groups. Conclusion Both Ct MOMP and HPV6b L1 protein specific cellular immune response could be induced in BALB/c mice immunized with HPV6b L1/Ct MOMP multi-epitope chimeric plasmid, and the HPV6b L1 gene optimized by eukaryotic codon could significantly enhance the cellular immune response induced by Ct MOMP multi-epitope gene in BALB/c mice.

Study of Chlamydial Urogenital Infection and MOMP Gene Variants in Child- bearing Women in Beijing / 中华皮肤科杂志

Objective To investigate Chlamydia trachomatis infection and gene variants in child- bearing women in Beijing. Methods Endocervical samples were collected from 177 patients in a family planning clinic and a gynecologic clinic. C.trachomatis infection was screened with a fast diagnostic kit .Major outer membrane protein(MOMP) gene was amplified by a nested PCR . A 1.1kb amplified product was obtained and studied by restriction fragment length polymorphism analysis(RFLP). Variant domains of MOMP gene VS1,VS2,VS3,VS4 were sequenced with 373A automated sequencing system. Results C. trachomatis was found in 9 of 177 samples. Four samples were identified as F genotype, 3 E genotype and 2 D genotype. Variant domains of MOMP gene, VS1, VS2, VS3, VS4 were sequenced and the results were consistent with those of RFLP, however, mutations were found in MOMP gene in 4 samples. Conclusion C.trachomatis urogenital infection has been identified in a portion of child- bearing women in Beijing. C.trachomatis can be well genotyped with RFLP or gene sequencing of MOMP gene.