OBJECTIVE: To investigate the value of clinical application of simultaneous amplification and testing of RNA (SAT-RNA) for detecting Chlamydia trachomatis (CT) and Ureaplasma urealyticum (UU) by comparing with the polymerase chain reaction testing of DNA (PCR-DNA) method. METHODS: Specimens from both urethra swab and the first avoid urine which should be at least one hour after the previous urination were collected from 163 men who were scheduled for in vitro fertilization and embryo transfer (IVF-ET) treatment due to female factors at Center for Reproductive Medicine, Shengjing Hospital of China Medical University during the period of April 2016 to April 2017. Among the 163 men, 109 simultaneously provided semen that was collected after 3-7 days of sexual abstinence for the testing. Urine and semen specimens were detected for CT and UU with SAT-RNA, while urethra swab specimens were detected for CT and UU with standard PCR-DNA. Detection results of the SAT-RNA were compared with those of the PCR-DNA method. RESULTS: The positive rate of UU in the urethra swab detected with PCR-DNA and that of UU in the urine with SAT-RNA were 47.24% and 47.85%, respectively, and the coincidence rate was 93.25%. In addition, the positive and negative coincidence rates were 93.51% and 93.02%, respectively, and the concordance between the two methods was very good (Kappa=0.865). On the other hand, the positive rate of CT in the swab specimen tested with PCR-DNA was 3.07% and that of CT in urine with SAT-RNA was 4.29%, and the coincidence rate was 97.55%. Moreover, the positive and negative coincidence rates were 80.00% and 98.10%, respectively, and the concordance between the two methods was good (Kappa=0.654). Regarding SAT-RNA detection of UU in the urine and semen specimen of the 109 patients, the positive rates of UU in the urine and semen specimens were 50.46% and 44.95%, respectively; and the coincidence rate between the two specimens was 88.99%. In addition, the positive coincidence rate and the negative coincidence rate was 93.88% and 85.00%, respectively, and the concordance between the two specimens was good (Kappa=0.780). Similarly, SAT-RNA detection of CT in the urine and semen specimens showed the positive rate was 5.50% and 3.67%, respectively; and the two specimens showed 98.17% coincidence rate. The positive and negative coincidence rates were 100.00% and 98.10%, respectively, and the concordance was also good (Kappa=0.791). CONCLUSION SAT-RNA detection of CT and UU in the urine specimen showed good concordance with the PCR-DNA detection of CT and UU in the urethra swab specimen. In addition, the concordance was also good between the urine and semen specimens detected with SAT-RNA. These results indicate that, as a less invasive and equally accurate procedure, SAT-RNA may be more suitable for clinical application.
Chlamydia Infections/epidemiology* ; Chlamydia trachomatis/genetics* ; Female ; Humans ; Infertility, Male ; Male ; Neisseria gonorrhoeae/genetics* ; Polymerase Chain Reaction ; Ureaplasma urealyticum/genetics*

BACKGROUND: The bacterium Chlamydia trachomatis is one of the leading causes of sexually transmitted diseases worldwide. Since no simple and effective tool exists to diagnose C. trachomatis infections, we evaluated a novel point-of-care (POC) test, aQcare Chlamydia TRF kit, which uses europium-chelated nanoparticles and a time-resolved fluorescence reader. METHODS: The test performance was evaluated by comparing the results obtained using the novel POC testing kit with those obtained using a nucleic acid amplification test (NAAT), using 114 NAAT-positive and 327 NAAT-negative samples. RESULTS: The cut-off value of the novel test was 20.8 with a detection limit of 0.27 ng/mL. No interference or cross-reactivity was observed. Diagnostic accuracy showed an overall sensitivity of 93.0% (106/114), specificity of 96.3% (315/327), positive predictive value (PPV) of 89.8% (106/118), and negative predictive value (NPV) of 97.5% (315/323). The sensitivity of the novel test was much higher than that of currently available POC tests. Furthermore, the relative ease and short turnaround time (30 min) of this assay enables C. trachomatis-infected individuals to be treated without a diagnostic delay. CONCLUSIONS: This simple and novel test is a potential tool to screen a larger population, especially those in areas with limited resources.
Adult ; Aged ; Aged, 80 and over ; Chlamydia Infections/*diagnosis ; Chlamydia trachomatis/*genetics/isolation & purification ; DNA, Bacterial/chemistry/metabolism ; Europium/*chemistry ; Female ; Humans ; Male ; Metal Nanoparticles/*chemistry ; Middle Aged ; Point-of-Care Systems ; Reagent Kits, Diagnostic ; Real-Time Polymerase Chain Reaction ; Sensitivity and Specificity ; Young Adult

BACKGROUND: The participation of laboratories in external quality assessment (EQA) programs is required for the quality assurance of nucleic acid amplification of Chlamydia trachomatis. This study aimed to construct a new quality control (QC) material applicated in EQA of C. trachomatis PCR. METHODS: A QC material-HTB-SiHa cells transfected with a recombinant plasmid containing the cryptic plasmid sequence-was constructed for C. trachomatis PCR detection, and four different panels, each consisting of 4 positive samples with serial dilution of the constructed QC material and 1 negative sample, were distributed by the National Center for Clinical Laboratories among four groups of 275, 268, 317, and 304 participants across China from 2011 through 2012. A total of eight commercial kits were used for C. trachomatis PCR detection in participants. RESULTS: Nine laboratories reported false-positive results (0.9%). As the series dilution increased, the correct reporting of the data sets decreased; the lowest correct rate was 96.3% in the weakest positive samples (104 copies/mL). Eight laboratories reported false-positive results, and 42 laboratories reported false-negative results in the EQA detection of C. trachomatis. No significant differences were observed in the detection of the constructed C. trachomatis positive samples (97.9%, 98.5%, 100%, 98.5%; P=0.36) and negative samples (100%, 99.0%, 100%, 99.0%; P=0.764) using four commercial kits commonly used in China. CONCLUSIONS: The results of the EQA study indicated that the constructed material provides a noninfectious, stable control material with sufficient volume for PCR detection of C. trachomatis.
Cell Line ; Chlamydia Infections/diagnosis ; Chlamydia trachomatis/*genetics ; DNA, Bacterial/*analysis ; False Negative Reactions ; Humans ; Laboratories/*standards ; Plasmids/genetics/*metabolism ; Polymerase Chain Reaction/*standards ; Quality Control ; Reagent Kits, Diagnostic

OBJECTIVETo review the common genotyping techniques of Chlamydia trachomatis in terms of their principles, characteristics, applications and limitations.

DATA SOURCESData used in this review were mainly from English literatures of PubMed database. The search terms were "Chlamydia trachomatis" and "genotyping". Meanwhile, data from World Health Organization were also cited.

STUDY SELECTIONOriginal articles and reviews relevant to present review's theme were selected.

RESULTSDifferent genotyping techniques were applied on different occasions according to their characteristics, especially in epidemiological studies worldwide, which pushed the study of Chlamydia trachomatis forward greatly. In addition, summaries of some epidemiological studies by genotyping were also included in this work for reference and comparison.

CONCLUSIONSA clear understanding of common genotyping techniques could be helpful to genotype C. trachomatis more appropriately and effectively. Furthermore, more studies on the association of genotypes of Chlamydia trachomatis with clinical manifestations should be performed.

Chlamydia trachomatis ; genetics ; Genotype ; Multilocus Sequence Typing ; Polymerase Chain Reaction ; Tandem Repeat Sequences ; genetics

Chlamydia trachomatis ; genetics ; Chlamydiaceae Infections ; diagnosis ; genetics ; Genes, Bacterial ; genetics ; Genes, Viral ; genetics ; Gonorrhea ; diagnosis ; genetics ; Herpes Simplex ; diagnosis ; genetics ; Herpesvirus 2, Human ; genetics ; Humans ; Multiplex Polymerase Chain Reaction ; Neisseria gonorrhoeae ; genetics ; Ureaplasma Infections ; diagnosis ; genetics ; Ureaplasma urealyticum ; genetics

OBJECTIVETo clone the plasmid protein pORF8 of Chlamydia trachomatis and localize its expression in Chlamydia-infected cells.

METHODSpORF8 gene was amplified and cloned into pGEX-6p vector, and the pORF8 fusion protein was expressed in E.coli XL1 Blue. After purification with glutathione-conjugated agarose beads, the pORF8 fusion protein was used to immunize BALB/c mice to generate polyclonal antibodies against pORF8 protein. The antibodies obtained were used to localize the plasmid protein pORF8 in Chlamydia-infected cells with immunofluorescence assay (IFA).

RESULTSThe pORF8 gene 744 bp in length was successfully cloned and the GST fusion protein with a relative molecular mass of 54 000 was obtained. The cellular distribution pattern of the plasmid protein pORF8 was similar to that of the major outer membrane protein (MOMP), a known C. trachomatis inclusion body protein, but not to that of chlamydial protease-like activity factor (CPAF, a secreted protein).

CONCLUSIONThe plasmid protein pORF8 is localized on the bacterial organism as an inclusion body protein in C. trachomatis-infected cells. The cellular location of pORF8 protein can potentially provide important insights into the pathogenesis of C. trachomatis.

Animals ; Antibodies ; immunology ; Bacterial Proteins ; biosynthesis ; genetics ; Chlamydia Infections ; metabolism ; Chlamydia trachomatis ; chemistry ; genetics ; metabolism ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; HeLa Cells ; Humans ; Mice ; Mice, Inbred BALB C ; Plasmids ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

Lymphocytic ascites with low serum-ascites albumin gradient (SAAG) are observed mainly in tuberculous peritonitis, peritoneal carcinomatosis, and pancreatic disease. However, pelvic inflammatory disease (PID) induced generalized peritonitis causing diffuse ascites has been rarely described. We report a 26-year old female patient, who was diagnosed as generalized peritonitis with diffuse ascites due to Chlamydia trachomatis infection. Gynecologic examination did not show the clue of PID and in the analysis of ascites, low SAAG, predominant lymphocyte count and high level of adenosine deaminase were noted. Although the best impression was tuberculous peritonitis on the base of these findings, the laparoscopic finding was consistent with PID and the PCR for C. trachomatis infection in cervical swab was positive. This case suggests that C. trachomatis peritonitis should be considered as a rare cause of low SAAG and lymphocytic ascites in sexually active women and should be intensively evaluated including laparoscopic examination.
Adult ; Anti-Bacterial Agents/therapeutic use ; Ascites/diagnosis/metabolism/therapy ; Ascitic Fluid/chemistry ; Cephalosporins/therapeutic use ; Chlamydia Infections/complications/*diagnosis/drug therapy ; Chlamydia trachomatis/genetics/*isolation & purification ; Diagnosis, Differential ; Female ; Humans ; Laparoscopy ; Peritonitis/*diagnosis/etiology/radiography ; Peritonitis, Tuberculous/diagnosis ; Serum Albumin/metabolism ; Tomography, X-Ray Computed

OBJECTIVETo investigate the antigenicity of recombinant Chlamydia trachomatis (Ct) OmcBc protein and search for the new target for early diagnosis of Chlamydia infection and Chlamydia vaccine development.

METHODSThe C fragment of OmcB encoding the amino acids from T270 to T553 was amplified from Chlamydia serovar D genomic DNA. The pGEX-6p-Ct OmcBc expression plasmid was constructed and transformed into E.coli XL-1blue. The expression of recombinant Ct OmcBc protein was induced by IPTG. Serum samples were collected from 120 patients with urogenital Chlamydia infection. The antiserum samples were collected from 7 New Zealand white rabbits and 5 Balb/C mice immunized subcutaneously and intraperitoneally with Ct serovar D inactivated EB, respectively, and from 9 Balb/C mice intranasally infected with Ct serovar D live EB. The anti-Chlamydia specific antibody were titrated by an immunofluorescence assay (IFA). The reactivity of the recombinant OmcBc protein with all the above antisera was detected by ELISA.

RESULTSThe pGEX-6p-Ct OmcBc expression plasmid was successfully constructed. DNA sequencing showed that the inserted OmcBc was about 852 bp, encoding a protein with 284 amino acids. The expression of the recombinant GST-OmcBc was induced by IPTG, producing a fusion protein with a molecular weight of about 57 kD. The titer of the specific antibodies to Chlamydia in all the antisera was high. ELISA results showed strong reactivities of the recombinant GST-OmcBc fusion protein with all the above antisera.

CONCLUSIONSOmcBc protein is an immunodominant protein of Chlamydia. The recombinant GST-OmcBc with strong antigenicity may provide a basis for further study of early diagnosis of chlamydia infection and development of Chlamydia vaccine.

Animals ; Antibodies, Bacterial ; blood ; Antigens, Bacterial ; immunology ; metabolism ; Bacterial Outer Membrane Proteins ; genetics ; immunology ; Chlamydia trachomatis ; genetics ; immunology ; metabolism ; Cloning, Molecular ; Genes, Bacterial ; Humans ; Immune Sera ; immunology ; Mice ; Mice, Inbred BALB C ; Plasmids ; Rabbits

OBJECTIVETo construct a recombinant eukaryotic expression plasmid pcDNA3.1-Ct MOMP168 including Ct MOMP multi-epitopes gene, and evaluate the Ct MOMP-specific humoral and cellular immune response induced by pcDNA3.1-Ct MOMP168 in BALB/c mice.

METHODSRecombinant plasmid pcDNA3.1-Ct MOMP168 including Ct MOMP multi-epitopes gene was constructed. Then, BALB/c mice were randomly assigned to receive (intramuscular injection) either pcDNA3.1-Ct MOMP168 or pcDNA3.1 or PBS (n = 12, 100 microg/time per mouse), and the same immunization schedule was repeated for the third time at 2 week intervals. The titers of anti-Ct MOMP antibody and its antibody subtypes in sera, the cytotoxicity of Ct MOMP-specific cytotoxic T lymphocyte (CTL) in spleen, and the level of cytokine (IFN-gamma, IL-4, IL-10)-producing CD3(+) T cells in spleen were detected by ELISA, LDH release assays and intracellular cytokine staining-fluorescence activated cell sorter (ICS-FACS), respectively.

RESULTSThe recombinant plasmid pcDNA3.1-Ct MOMP168 was able to induce Ct-specific antibody response (A(490) = 0.973 +/- 0.136; serum titer was 1:1000) as compared with pcDNA3.1 (A(490) = 0.180 +/- 0.025) and PBS (A(490) = 0.110 +/- 0.015), and the major antibody subtype was IgG2a with statistical significance (F = 106.884, P < 0.05). When the ratio of effector cells and target cells reached to 50:1, the activity of cytotoxic T-lymphocyte in pcDNA3.1-Ct MOMP168 immunized mice (41.71% +/- 8.34%) was significantly higher (F = 22.315, P < 0.05) than that in pcDNA3.1 immunized mice (18.40% +/- 3.45%) and PBS immunized mice (14.50% +/- 2.42%). The levels of CD3(+) IFN-gamma(+) T cells in pcDNA3.1-Ct MOMP168 immunized mice (1.15% +/- 0.16%) were significantly higher (F = 99.638, P < 0.05) than that in pcDNA3.1 immunized mice (0.12% +/- 0.08%) and PBS immunized mice (0.09% +/- 0.03%), while the significant difference in the levels of IL-4(+) CD3(+) T cells and IL-10(+) CD3(+) T cells was not observed (F = 0.886 and 1.112, P > 0.05) between pcDNA3.1-Ct MOMP168 immunized mice (0.13% +/- 0.08% and 0.14% +/- 0.08%) and pcDNA3.1 (0.07% +/- 0.05% and 0.13% +/- 0.06%) or PBS immunized mice (0.08% +/- 0.04% and 0.07% +/- 0.04%).

CONCLUSIONIn BALB/c mice, the recombinant plasmid pcDNA3.1-Ct MOMP168 might induce not only the generation of Ct-specific antibody, but also the high level of Ct MOMP-specific CD3(+) IFN-gamma(+) T cells.

Animals ; Bacterial Outer Membrane Proteins ; genetics ; immunology ; Bacterial Vaccines ; immunology ; Chlamydia trachomatis ; genetics ; immunology ; Immunization ; Male ; Mice ; Mice, Inbred BALB C ; Porins ; genetics ; immunology ; T-Lymphocytes, Cytotoxic ; immunology

OBJECTIVETo study the role of pathogenic microorganisms commonly associated with chronic eye disease, including Chlamydia psittaci, Chlamydia trachomatis, Chlamydia pneumoniae, herpes simplex virus (HSV) type 1 and type 2, and adenovirus type 8 and type 19, in the development of primary ocular adnexal mucosa-associated lymphoid tissue (MALT) lymphoma in Chinese patients.

METHODSSixty-eight archival cases of primary ocular adnexal lymphoproliferative lesions, including 38 cases of MALT lymphoma, 3 cases of non-MALT lymphoma and 27 cases of chronic inflammation, were enrolled into the study. DNA was extracted from the paraffin-embedded tissue samples. The presence of DNA of C. psittaci, C. trachomatis, C. pneumoniae, HSV type 1, HSV type 2, adenovirus type 8 and adenovirus type 19 were analyzed by multiplex touchdown enzyme time-release polymerase chain reaction (TETR-PCR).

RESULTSAll of the specimens yielded PCR products of over 100 base pairs and were thus suitable for TETR-PCR screening of infectious agents. The prevalence of DNA of C. psittaci, C. trachomatis and adenovirus type 19 were 0 in MALT lymphoma, non-MALT lymphoma and chronic inflammation. There were 2 cases positive for C. pneumoniae DNA, amongst the 38 cases of MALT lymphoma studied (5.3%, 2/38). HSV type 1, HSV type 2 and adenovirus type 8 DNA was found in each of the 3 patients with chronic inflammation.

CONCLUSIONThe study indicates that C. psittaci, C. trachomatis, C. pneumoniae, HSV type 1, HSV type 2, adenovirus type 8 and adenovirus type 19 probably play little role in the pathogenesis of ocular adnexal MALT lymphoma in Chinese patients.

Adenovirus Infections, Human ; virology ; Adenoviruses, Human ; genetics ; isolation & purification ; Chlamydia Infections ; microbiology ; Chlamydia trachomatis ; genetics ; isolation & purification ; Chlamydophila Infections ; microbiology ; Chlamydophila pneumoniae ; genetics ; isolation & purification ; Chlamydophila psittaci ; genetics ; isolation & purification ; DNA, Bacterial ; analysis ; DNA, Viral ; analysis ; Eye Infections ; microbiology ; virology ; Eye Neoplasms ; microbiology ; virology ; Herpes Simplex ; virology ; Herpesvirus 1, Human ; genetics ; isolation & purification ; Herpesvirus 2, Human ; genetics ; isolation & purification ; Humans ; Lymphoma, B-Cell, Marginal Zone ; microbiology ; virology ; Psittacosis ; microbiology