Objective: To develop a sensitive, specific and simple method for detection of extremely low numbers of T. pallidum in clinical specimens, as a significant addition to the serologic tests for syphilis diagnosis. Metho...Objective: To develop a sensitive, specific and simple method for detection of extremely low numbers of T. pallidum in clinical specimens, as a significant addition to the serologic tests for syphilis diagnosis. Methods: Double-tube nested PCR(DN-PCR) and single-tube nested PCR(SN-PCR) assays were performed to amplify specific fragments of the DNA poly-merase I gene(polA) of T. pallidum. Sensitivity and specificity of the two PCR assays were tested. Eighty-six whole blood specimens from persons with suspected syphilis were detected by the two nested PCR methods. The TPPA test was used as a comparison for detecting syphilis in sera from corresponding patients. Results: Only specific amplicons could be obtained during amplification of the T. pallidum polA gene and the detection limit was approximately 1 organism when analyzed on gel by the two PCR methods. Of 86 clinical specimens, 62 were positive by TPPA. Of these, 54 and 51 were positive by the DN-PCR and SN-PCR, respectively, which does not represent a statistically significant difference between the two PCR tests. Of 24 TPPA-negative specimens, 5 were positive by both DN-PCR assay and SN-PCR assay. Conclusion: The SN- polA PCR method is extremely sensitive, specific and easy to perform for detecting low numbers of T. pallidum in clinical blood specimens as a complementary to serology for syphilis diagnosis.展开更多
Objectives： To clone and express Tp0453 outer membrane protein of Treponema pallidum, and to evaluate its significance in the serodiagnosis of syphilis. Methods： The immuno-dominant epitope of Tp0453 gene was amplif...Objectives： To clone and express Tp0453 outer membrane protein of Treponema pallidum, and to evaluate its significance in the serodiagnosis of syphilis. Methods： The immuno-dominant epitope of Tp0453 gene was amplified from the complete genome of T.pallidum by polymerase chain reactions （PCR）, subcloned into the expression vector Pqe32 to generate recombinant plasmid Pqe32/Tp0453, and was then expressed in E. coli M15. The fusion protein was purified with Ni-NTA affinity purification. Indirect ELISA was developed to detect human serum IgG antibody to T. pallidum. Results： The recombinant Tp0453 protein was successfully expressed and purified. The recombinant protein had a molecular weight of approximately 32KDa.Indirect ELISA to the recombinant protein was developed.Sixty control sera were tested by ELISA and yielded a sensitivity of 100% （30/30） and a specificity of 100% （30/30）. While testing for T. pallidum in human sera, the sensitivity and specificity of ELISA were 96.8% and 100%, respectively, when compared with TPPA test results. The concordance of results between the ELISA test and the TPPA test was 98.2%. Conclusion： The recombinant Tp0453 outer membrane protein elicited a strong immunoreaction to anti-T.pallidum IgG antibody and has great potential use in ELISA for the serodiagnosis of syphilis.展开更多
Objectives: To develop a multi-nested polymerase chain reaction in an assay to detect early Treponema pallidum and Haemophilius ducreyi DNA in the swabs of genital ulcers. Methods: Four pairs of outer and inner primer...Objectives: To develop a multi-nested polymerase chain reaction in an assay to detect early Treponema pallidum and Haemophilius ducreyi DNA in the swabs of genital ulcers. Methods: Four pairs of outer and inner primers, specific to the basic membrane protein gene of Treponema pallidum and to the 16s rRNA gene of H ducreyi were synthesized. The multi-nested PCR was developed and applied to detect Treponema pallidum and Haemophilus dicreyi in clinical swabs. Result: The two samples of standard strains of Haemophilus ducreyi and one Treponema pallidum were amplified and showed 309-bp rRNA gene of Haemophilus ducreyi and 506-bp DNA of Treponema palidum, respectively. Out of 51 samples of genital ulcer detected, 29 showed Treponema pallidum positive product and no Haemophilus ducreyi DNA was found. Conclusion: The multi-nested PCR for Treponema pallidum and Haemophilus ducreyi could be useful for early detection and distinguishing diagnosis between syphilis and chancroid.展开更多
Objective To assess the sensitivity, specificity, and feasibility of 4 recombinant Treponema pallidum antigenbased rapid tests in the diagnosis of syphilis. Methods A total of 970 outpatients were selected from the S...Objective To assess the sensitivity, specificity, and feasibility of 4 recombinant Treponema pallidum antigenbased rapid tests in the diagnosis of syphilis. Methods A total of 970 outpatients were selected from the Sexually Transmitted Diseases Centre of Peking Union Medical College Hospital. Venous blood was collected and serum was extracted. T. paUidum antibodies in whole blood, anticoagulant whole blood, and serum were detected using 4 recombinant T. pallidum antigen-based rapid tests. T. pallidum haemagglutination test （TPHA） was considered as the gold standard for the detection of T. pallidum specific antibodies in serum. The sensitivities and specificities of four methods were analyzed. Results The sensitivities and specificities of Abbott Determine Syphilis TP test, SD-BIOLINE Syphilis 3.0 test, VISITECT-SYPHILIS test, and Syphicheck-WB test for serum specimens were 100% and 98. 9%, 95.7% and 98.0%, 94.6% and 98.2%, 68.1% and 98.9% ; for whole blood were 74. 1% and 99. 5%, 87.9% and 99.4% , 73.2% and 99.7%, 64. 7% and 99.7%. The observed sensitivities of the 4 rapid diagnosis tests were not significantly different with TPHA （ P 〉 0.05 ）. Conclusions The 4 rapid tests show good performance and characteristics in the diagnosis of syphilis. Furthermore, they are more sensitive for serum specimens than whole blood.展开更多
To construct the recombinant plasmid of eukaryotic expression containing Tp92 gene from Treponema pallidum and study its immunogenicity in New Zealand white rabbits. Tp92 gene was amplified from the genomic DNA of T. ...To construct the recombinant plasmid of eukaryotic expression containing Tp92 gene from Treponema pallidum and study its immunogenicity in New Zealand white rabbits. Tp92 gene was amplified from the genomic DNA of T. pallidum by polymerase chain reaction (PCR) and subcloned into appropriate site of pcDNA3.1(+) vector. After identification by sequencing and restrictive enzyme digestion, the recombinant plasmid was transfected into HeLa cells using liposome, and the expressed protein was identified by immunocytochemistry and Western blotting. After verifying that the Tp92 antigen gene fragment could be expressed in HeLa cells, 100?μg of recombinant plasmids [pcDNA3.1(+)-Tp92], 100 μg of control plasmids [pcDNA3.1(+)] or 0.5 ml PBS buffer were administered in 3 groups of New Zealand white rabbits (6 rabbits/group), and the booster immunizations were employed at 2-week interval for 3 times. ELISA assay was used for the quantitative detection of the specific antibody in the sera of rabbits, and the proliferation response of spleen cells was detected by MTT assay. It was found that the target gene Tp92 segment about 2103 bp was obtained, and the DNA sequence of Tp92 gene constructed in pcDNA3.1 (+) vector was consistent with the published nucleotide sequence. The homologies of the nucleotide and putative amino acid sequences of Tp92 gene between T.pallidum subsp. pallidum Nichols and various pathogenic treponeme strains were 95.5%-100%. The analysis of immunocytochemistry and Western blotting showed that Tp92 gene segment constructed in pcDNA3.1(+) vector could express a fusion protein with a calculated molecular mass of 77 kDa in HeLa cells and the expressed protein could react with positive blood serum from syphilis patient. The specific antibody IgG titers were observed and the highest titer was 1∶1024 in rabbits after 3 times with pcDNA3.1(+)-Tp92. The proliferation response of spleen cells were significantly higher than that of rabbits injected with pcDNA3.1(+) ( P <0.05). The successful expression of the eukaryotic expression plasmid of Tp92 gene from T. pallidum was obtained in eukaryotic system and strong responses of humoral and cellular immunity was evoked by DNA vaccine of pcDNA3.1(+)-Tp92 in rabbits thus establishing a solid basis for the future studies in the biological activities and for the development of the syphilis DNA vaccine.展开更多
To clone and express the recombinant outer membrane protein Tp0453 of Treponema pallidum and to analyze the immuno-reactivity and immunogenicity of the expressed protein, the immuno-dominant epitope of the Tp0453 was ...To clone and express the recombinant outer membrane protein Tp0453 of Treponema pallidum and to analyze the immuno-reactivity and immunogenicity of the expressed protein, the immuno-dominant epitope of the Tp0453 was amplified from the complete genome of T.pallidum by PCR, subcloned into expression vector pQE32 to generate the recombinant plasmid pQE32/Tp0453, then expressed in E.coli M15 and analyzed by SDS/PAGE and Western blotting. The fusion protein expressed was purified with Ni-NTA affinity chromatography. Its immuno-reactivity was assayed by indirect ELISA, and the immunogenicity was determined by immunization with this fusion protein in New Zealand rabbits. In the present study, a fusion protein of molecular weight about 32 kDa was obtained. As demonstrated by Western blotting, the recombinant protein could react specifically with positive IgG sera of patients with syphilis, and the antibodies against T.pallidum in human sera were successfully detected by indirect ELISA. Both the sensitivity and specificity of ELISA based on the Tp0453 fusion protein as were 100% (30/30) when detected with control sera. In comparison with the results of IgG ELISA with those of TPPA. It was found that the sensitivity of ELISA was 96.8% and the specificity was 100%. The difference of ELISA and TPPA was not significant, and the concordance of results between ELISA and TPPA was 98.2%. In addition, specific humoral responses could be elicited by immunization with the recombinant fusion protein in New Zealand rabbits with a specific antibody titer of 1∶1280 after 3 successive doses of immunization. These results demonstrate that the expressed recombinant fusion protein shows excellent immuno-competence and provide foundation to develop a quick diagnostic kid applied to detect the presence of T.pallidum infections.展开更多
The Gpd gene was amplified from the genomic DNA of Treponema the appropriate site of pcDNA3.1 （ ＋ ） vector. The expression of pcDNA3. I was tested with Western blotting and technology of immunoeytochemisty. New mun...The Gpd gene was amplified from the genomic DNA of Treponema the appropriate site of pcDNA3.1 （ ＋ ） vector. The expression of pcDNA3. I was tested with Western blotting and technology of immunoeytochemisty. New munized with the eukaryotic expression recombinant pcDNA3, 1 （ ＋ ）-Gpd pallidum and cloned into （ ＋ ）-Gpd in Hel,a cells Zealand rabbits were imA fusion protein of C, pd with 4.1 kDa has been effectively expressed in HeLa cells, which were detected bv Western blotting and the immunocytochemistry techniques. The New Zealand rabbits were able to elicit the specific antibody after immunization with the nucleic acid vaccine. The antibody titer could reach as high as 1 ： 1024 after 2 weeks of the third injection; and the splenocytes proliferated evidently due to the Gpd protein stimulation. Both the antibody titer and the splenocytes proliferation were higher substantially than those of controls （ P 〈 0.01 ）. All above data will contribute to an experimental basis of further study of the biological function of Gpd protein as well as DNA vaccine for syphilis.展开更多
Objective: To evaluate the clinical application of multiplex PCR inthe detection of Treponema pallidum, Herpes simplex virus (HSV), andHaemophilus ducreyi. Method: Three standard strains were used to set up a multiple...Objective: To evaluate the clinical application of multiplex PCR inthe detection of Treponema pallidum, Herpes simplex virus (HSV), andHaemophilus ducreyi. Method: Three standard strains were used to set up a multiplexPCR (MPCR) for detecting syphilis, herpes genitalis, and chancroidsimultaneously. Samples from 122 patients with genital ulcer disease(GUD) were subjected to MPCR and the results were compared withthose of dark-field microscopy and TP serology, HSV antigen ELISA,and H. ducreyi culture. Results: In the 122 patients with GUD, MPCR identified 34 casesof T. pallidum infection, 40 cases of HSV infection, and 2 cases of mixedinfection of T. pollidum and herpes. No positive results of H. ducreyiwere found. The sensitivity of MPCR to T. pallidum and herpes was100% and 93.3%, respectively. The sensitivities of dark-field mi-croscopy and TP serology, HSV antigen ELISA, and H. ducreyi cul-ture was 35.3%, 50% and 100%, respectively. Conclusion: MPCR showed a relatively higher sensitivity for T.pallidum as compared with the routine techniques. Although its sensi-tivity for HSV was not as good as that of antigen ELISA, it also yieldeda high detection rate. MPCR can detect more than one pathogen. It issimple, quick, sensitive, and suitable for clinical use or epidemiologicalinvestigation.展开更多
Objective: To construct the recombinant plasmid containing Glycerophosphodiester phosphodiesterase (Gpd) gene from Treponema pallidum and transfect it into Hela cells to express the encoded outer membrane protein. Met...Objective: To construct the recombinant plasmid containing Glycerophosphodiester phosphodiesterase (Gpd) gene from Treponema pallidum and transfect it into Hela cells to express the encoded outer membrane protein. Methods: The Gpd gene was amplified from the genomic DNA of T.pallidum by polymerase chain reaction (PCR) and inserted into cloning vector pUCm-T. The inserted Gpd gene was subcloned into the appropriate site of pcDNA3.1(+) vector. After identification by sequencing and restrictive enzymes digestion, the recombinant plasmid was transfected into Hela cells using liposomes. The expressed protein was identified by immunocytochemistry and Western blot. Results: The target Gpd gene segment was approximately 1059bp. The DNA sequence of the Gpd gene contained in the pcDNA3.1(+) vector was consistent with the published nucleotide sequence. The homology of the nucleotide and putative amino acid sequences of the Gpd gene between T. pallidum subsp. pallidum Nichols and various pathogenic treponemal strains ranged from 98% to 100%. Immunocytochemistry and Western blot analysis showed that the constructed Gpd-pcDNA3.1(+) vector expressed a fusion protein with a calculated molecular mass of 41KDa in Hela cells and that the expressed protein reacted with the sera from syphilis patients. Conclusion: The successful construction and expression of the eukaryotic expression plasmid of the Gpd gene from T.pallidum provide a promising tool to further study the biological activity of T.pallidum and develop a DNA vaccine for syphilis.展开更多
Objective: To obtain recombinant Treponema pallidumsubsp pallidum (TP 17KD) lipoprotein in large quantities byamplification and to further purify antigens for laboratorydiagnosis of syphilis and development of a syphi...Objective: To obtain recombinant Treponema pallidumsubsp pallidum (TP 17KD) lipoprotein in large quantities byamplification and to further purify antigens for laboratorydiagnosis of syphilis and development of a syphilis vaccine. Method: The Tpp17 lipoprotein gene was amplified fromthe TP(strain Nichols), and then it was recombinated into aplasmid pMAL-2c and cloned within E coli l2-TB1. The hostbacteria containing recombinant plasmids were induced withIPTG. The Tpp 17KD lipoprotein gene was amplified by us-ing PCR and positive clones were screened with double diges-tion and PCR. Recombinant plasmids were transformed intoE. coli and the E coli carrying recombinant plasmids wereinduced. The expression of TP 17KD was detected by sodiumdedecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and immunoblot. Results:Gel staining with Coomassie blue G-250 showedthat the induced E coli carrying recombinant plasmid couldproduce 60KD fusion protein at high levels. Gel scanningshowed that 17KD protein expression in E coli accounted for10% of total cellular protein. The recombinant protein antigenreacted with the sera of syphilis patients. Conclusion: Our study lays a cornerstone for developingnew techniques of laboratory diagnosis for syphilis and newvaccines. Preliminary clinical application showed that thefusion protein could be used for the diagnosis of syphilis.展开更多
Objective:To apply a regression equation to calculate the gray zone range for detecting Treponema pallidum antibody(TP-Ab)in blood samples using the ARCHITECT i2000SR fully automated microparticle chemiluminescence an...Objective:To apply a regression equation to calculate the gray zone range for detecting Treponema pallidum antibody(TP-Ab)in blood samples using the ARCHITECT i2000SR fully automated microparticle chemiluminescence analyzer.Methods:A total of 180 blood samples initially screened for syphilis antibodies in our hospital were collected,which were tested using both the Treponema pallidum chemiluminescent microparticle immunoassay(TP-CMIA)on the ARCHITECT i2000SR electrochemical luminescence analyzer and the Treponema pallidum particle agglutination assay(TPPA).The optimal regression equation was established based on the TPPA cut-off index(COI)values and positive predictive probability,which was then used to determine the gray zone range for TP-CMIA.Results:This study analyzed 180 serum samples using a binary logistic regression model to evaluate the predictive value of COI values determined by the TP-CMIA method for syphilis.The Hosmer-Lemeshow test yielded P=0.332,further confirming the model's goodness-of-fit.The COI value was identified as a sensitive predictor of syphilis,with the cubic curve model demonstrating optimal fitting performance(R^(2)=0.925).When the positive predictive probability reached 95%,the corresponding COI value was 5.353,indicating that samples with COI values exceeding 5.353 had a true-positive probability>95%.Consequently,the gray zone range for TP-CMIA COI values was defined as 1.000-5.353,with values below this range classified as a false-positive interval.The model accurately classified 98.7%of syphilis cases and 87.0%of non-syphilis cases,achieving an overall correct classification rate of 97.2%.Conclusion:The gray zone range for COI values of TP-Ab detected by the TP-CMIA method(ARCHITECT i2000SR)was established as 1.000-5.353 using a regression equation,with samples showing COI values≤5.353 carrying a potential false-positive risk.展开更多
Treponema is a Gram-negative anaerobic bacterium,among which the pathogenic Treponema can cause various diseases,such as venereal syphilis(Treponema pallidum),yaws(Treponema carateum),and oral diseases(Treponema denti...Treponema is a Gram-negative anaerobic bacterium,among which the pathogenic Treponema can cause various diseases,such as venereal syphilis(Treponema pallidum),yaws(Treponema carateum),and oral diseases(Treponema denticola and Treponema medium).Although different from conventional lipopolysaccharides,the extracellular glycoconjugate of Treponema may still be a potential antigen and provide a candidate for vaccine development.Hence,we completed the first chemical synthesis of Treponema medium ATCC 700293 tetrasaccharide precursor containing L-ornithine(L-Orn)and D-aspartic acid(D-Asp)derivatives.The efficiency of non-reducing end disaccharide formation has been improved by optimizing the assembly of the protecting groups in the donors and acceptors.Our[3+1]glycosylation strategy attempted to reduce the length of the acceptor to increase the nucleophilicity of the hydroxyl group,thereby improving the efficiency of synthesizing the target tetrasaccharide.The L-Orn derivative was introduced at the final stage due to its influence on the glycosylation stereospecificity and efficiency.Therefore,the successful introduction of two amino acid derivatives and the synthesis of a tetrasaccharide precursor with complex functional-group modifications have provided valuable insights for synthesizing other complex bacterial glycans.展开更多
BACKGROUND The liver is the most common site of digestive system tumor metastasis,but not all liver metastases can be traced back to the primary lesions.Although it is unusual,syphilis can impact the liver,manifesting...BACKGROUND The liver is the most common site of digestive system tumor metastasis,but not all liver metastases can be traced back to the primary lesions.Although it is unusual,syphilis can impact the liver,manifesting as syphilitic hepatitis with inflammatory nodules,which might be misdiagnosed as metastasis.CASE SUMMARY This case report involves a 46-year-old female who developed right upper abdominal pain and intermittent low fever that persisted for more than three months.No definitive diagnosis of a tumor had been made in the past decades,but signs of multiple liver metastases were recognized after a computed tomo-graphy scan without evidence of primary lesions.With positive serological tests for syphilis and a biopsy of the liver nodules,a diagnosis of hepatic syphilis was made and confirmed with follow-up nodule reduction after anti-syphilis therapy.CONCLUSION Clinicians must be aware of the possibility that syphilis can cause hepatic inflam-matory masses,especially when liver metastasis is suspected without evidence of primary lesions.A definitive diagnosis should be established in conjunction with a review of the patient’s medical history for accurate therapeutic intervention.展开更多
The tissue destruction characteristic of syphilis infection may be caused by inflammation due to Treponema pallidum and the ensuing immune responses to the pathogen.T.pallidum membrane proteins are thought to be poten...The tissue destruction characteristic of syphilis infection may be caused by inflammation due to Treponema pallidum and the ensuing immune responses to the pathogen.T.pallidum membrane proteins are thought to be potent inducers of inflammation during the early stages of infection.However,the actual membrane proteins that induce inflammatory cytokine production are not known,nor are the molecular mechanisms responsible for triggering and sustaining the inflammatory cascades.In the present study,Tp0751 recombinant protein from T.pallidum was found to induce the production of proinflammatory cytokines,including TNF-α,IL-1βand IL-6,in a THP-1 human monocyte cell line.The signal transduction pathways involved in the production of these cytokines were then further investigated.No inhibition of TNF-a,IL-1β,or IL-6 production was observed following treatment with the SAPK/JNK specific inhibitor SP600125 or with an ERK inhibitor PD98059.By contrast,anti-TLR2 mAb,anti-CD14 mAb,and the p38 inhibitor SB203580 significantly inhibited the production of all three cytokines.In addition,pyrrolidine dithiocarbamate (PDTC),a specific inhibitor of NF-κB,profoundly inhibited the production of these cytokines.Tp0751 treatment strongly activated NF-κB,as revealed by Western blotting.However,NF-κB translocation was significantly inhibited by treatment with PDTC.These results indicated that TLR2,CD14,MAPKs/p38,and NF-κB might be implicated in the inflammatory reaction caused by T.pallidum infection.展开更多
Background Tp15, Tp17, Tp45, and Tp47 are outer-membrane proteins found in Treponema pallidum, the etiologic agent of syphilis. These proteins are potent antigens and are potential markers for the serological detectio...Background Tp15, Tp17, Tp45, and Tp47 are outer-membrane proteins found in Treponema pallidum, the etiologic agent of syphilis. These proteins are potent antigens and are potential markers for the serological detection of syphilis. The present study analyzed antibodies to these protein antigens （TP-IgM and TP-IgG） in human serum and investigated the expression of these antibodies during different stages of syphilis. Methods Serum samples were collected from 69 subjects （male 45, female 24） diagnosed with syphilis and analyzed by Western blotting for the expression of IgM and IgG against the four protein antigens. Expression levels of the target antibodies were compared during the same stage of syphilis as well as between different stages of this disease. Results In subjects with primary syphilis, the positive rate of Tp45 IgM was higher than that of other TP-IgM. Tp15 IgM was detected only in subjects with tertiary syphilis. Similarly, the seroprevalence of Tp45 IgG in primary syphilis was higher than for other TP-IgG. No target TP-IgM was detected in subjects with latent syphilis. In subjects with secondary syphilis, the expression level of Tp15 IgG （138.73±20.16） was higher than for other target TP-IgG. In subjects with tertiary syphilis, all target TP-IgG were detected. In subjects with tertiary or latent syphilis, the expression levels of Tp45 IgG （121.33±11.04 and 110.10±40.19, respectively） were higher than those of other target TP-IgG. The expression levels of all Tp-lgM were similar before or after anti-syphilis treatment. In comparison, the expression levels of all TP-IgG decreased compared with the pre-treatment levels, and this decrease was statistically significant （both P 〈0.05） for Tp17 IgG and Tp47 IgG. Conclusions After Treponema pallidum infection, Tp45 IgM appeared first and Tp15 IgM occurred during later stages. The positive rates of all TP-IgG increased with the duration of this disease. Anti-syphilis treatment reduced the expression levels of Tp17 IgG and Tp47 IgG. Larger-scale studies are required to further validate the value of Tp15, Tp17, Tp45, and Tp47 as markers for the early detection of primary and latent syphilis.展开更多
Background Treponema pallidum （T. pallidum） subsp, pallidum is the causative agent of syphilis. Analysis of recombinant antigens of T. pallidum led to the identification of potential candidate antigens for vaccine d...Background Treponema pallidum （T. pallidum） subsp, pallidum is the causative agent of syphilis. Analysis of recombinant antigens of T. pallidum led to the identification of potential candidate antigens for vaccine development and syphilis serodiagnosis. Tp0965 was predicted to be a membrane fusion protein and was found to be reactive with infected human sera in previous studies, but the results were controversial. In this research, the antigenicity and immunoreactivity of recombinant protein Tp0965 were assessed. Methods T. pallidum subsp, pallidum （Nichols strain） was propagated and isolated and the genomic DNA was extracted. The Tp0965 gene was amplified by polymerase chain reaction （PCR）. Then the recombinant protein Tp0965 was expressed in Escherichia coli and purified by nickel-nitrilotriacetic acid （Ni-NTA） purification system. The reactivities of protein Tp0965 were examined by immunoblot analysis and indirect enzyme-linked immunosorbent assay. The antisera against protein Tp0965 were obtained by immune rabbits and the immunogenicity of antisera were detected by indirect enzyme-linked immunosorbent assay. Results Recombinant protein Tp0965 was expressed successfully in vitro. Immunoblot assay showed that the recombinant protein Tp0965 could be recognized by human syphilitic sera of all stages. Indirect enzyme-linked immunosorbent assay showed there were only 4 of 74 human syphilitic sera that failed to show reactivity to recombinant antigen Tp0965, and lack of reactivity of Tp0965 to all 28 uninfected sera. A low titer of antiserum against Tp0965 in immune rabbits could be detected after the third time of immunization. Conclusions The recombinant antigen Tp0965 shows excellent sensitivity for the reactivity with sera from syphilitic individuals at all stages. The results also demonstrate a potential application for the serodiagnosis of syphilis.展开更多
文摘Objective: To develop a sensitive, specific and simple method for detection of extremely low numbers of T. pallidum in clinical specimens, as a significant addition to the serologic tests for syphilis diagnosis. Methods: Double-tube nested PCR(DN-PCR) and single-tube nested PCR(SN-PCR) assays were performed to amplify specific fragments of the DNA poly-merase I gene(polA) of T. pallidum. Sensitivity and specificity of the two PCR assays were tested. Eighty-six whole blood specimens from persons with suspected syphilis were detected by the two nested PCR methods. The TPPA test was used as a comparison for detecting syphilis in sera from corresponding patients. Results: Only specific amplicons could be obtained during amplification of the T. pallidum polA gene and the detection limit was approximately 1 organism when analyzed on gel by the two PCR methods. Of 86 clinical specimens, 62 were positive by TPPA. Of these, 54 and 51 were positive by the DN-PCR and SN-PCR, respectively, which does not represent a statistically significant difference between the two PCR tests. Of 24 TPPA-negative specimens, 5 were positive by both DN-PCR assay and SN-PCR assay. Conclusion: The SN- polA PCR method is extremely sensitive, specific and easy to perform for detecting low numbers of T. pallidum in clinical blood specimens as a complementary to serology for syphilis diagnosis.
基金Financially supported by Key grant from the Education Committee of Hunan Province (No. 02A046)
文摘Objectives： To clone and express Tp0453 outer membrane protein of Treponema pallidum, and to evaluate its significance in the serodiagnosis of syphilis. Methods： The immuno-dominant epitope of Tp0453 gene was amplified from the complete genome of T.pallidum by polymerase chain reactions （PCR）, subcloned into the expression vector Pqe32 to generate recombinant plasmid Pqe32/Tp0453, and was then expressed in E. coli M15. The fusion protein was purified with Ni-NTA affinity purification. Indirect ELISA was developed to detect human serum IgG antibody to T. pallidum. Results： The recombinant Tp0453 protein was successfully expressed and purified. The recombinant protein had a molecular weight of approximately 32KDa.Indirect ELISA to the recombinant protein was developed.Sixty control sera were tested by ELISA and yielded a sensitivity of 100% （30/30） and a specificity of 100% （30/30）. While testing for T. pallidum in human sera, the sensitivity and specificity of ELISA were 96.8% and 100%, respectively, when compared with TPPA test results. The concordance of results between the ELISA test and the TPPA test was 98.2%. Conclusion： The recombinant Tp0453 outer membrane protein elicited a strong immunoreaction to anti-T.pallidum IgG antibody and has great potential use in ELISA for the serodiagnosis of syphilis.
文摘Objectives: To develop a multi-nested polymerase chain reaction in an assay to detect early Treponema pallidum and Haemophilius ducreyi DNA in the swabs of genital ulcers. Methods: Four pairs of outer and inner primers, specific to the basic membrane protein gene of Treponema pallidum and to the 16s rRNA gene of H ducreyi were synthesized. The multi-nested PCR was developed and applied to detect Treponema pallidum and Haemophilus dicreyi in clinical swabs. Result: The two samples of standard strains of Haemophilus ducreyi and one Treponema pallidum were amplified and showed 309-bp rRNA gene of Haemophilus ducreyi and 506-bp DNA of Treponema palidum, respectively. Out of 51 samples of genital ulcer detected, 29 showed Treponema pallidum positive product and no Haemophilus ducreyi DNA was found. Conclusion: The multi-nested PCR for Treponema pallidum and Haemophilus ducreyi could be useful for early detection and distinguishing diagnosis between syphilis and chancroid.
基金Supported by the WHO project on rapid diagnosis of syphilis (RFA-SDI-2001-02)
文摘Objective To assess the sensitivity, specificity, and feasibility of 4 recombinant Treponema pallidum antigenbased rapid tests in the diagnosis of syphilis. Methods A total of 970 outpatients were selected from the Sexually Transmitted Diseases Centre of Peking Union Medical College Hospital. Venous blood was collected and serum was extracted. T. paUidum antibodies in whole blood, anticoagulant whole blood, and serum were detected using 4 recombinant T. pallidum antigen-based rapid tests. T. pallidum haemagglutination test （TPHA） was considered as the gold standard for the detection of T. pallidum specific antibodies in serum. The sensitivities and specificities of four methods were analyzed. Results The sensitivities and specificities of Abbott Determine Syphilis TP test, SD-BIOLINE Syphilis 3.0 test, VISITECT-SYPHILIS test, and Syphicheck-WB test for serum specimens were 100% and 98. 9%, 95.7% and 98.0%, 94.6% and 98.2%, 68.1% and 98.9% ; for whole blood were 74. 1% and 99. 5%, 87.9% and 99.4% , 73.2% and 99.7%, 64. 7% and 99.7%. The observed sensitivities of the 4 rapid diagnosis tests were not significantly different with TPHA （ P 〉 0.05 ）. Conclusions The 4 rapid tests show good performance and characteristics in the diagnosis of syphilis. Furthermore, they are more sensitive for serum specimens than whole blood.
基金This work was supported by Hunan Provincial Education Department (002A046) and Department of Public Health of Hunan province (B2003 085)
文摘To construct the recombinant plasmid of eukaryotic expression containing Tp92 gene from Treponema pallidum and study its immunogenicity in New Zealand white rabbits. Tp92 gene was amplified from the genomic DNA of T. pallidum by polymerase chain reaction (PCR) and subcloned into appropriate site of pcDNA3.1(+) vector. After identification by sequencing and restrictive enzyme digestion, the recombinant plasmid was transfected into HeLa cells using liposome, and the expressed protein was identified by immunocytochemistry and Western blotting. After verifying that the Tp92 antigen gene fragment could be expressed in HeLa cells, 100?μg of recombinant plasmids [pcDNA3.1(+)-Tp92], 100 μg of control plasmids [pcDNA3.1(+)] or 0.5 ml PBS buffer were administered in 3 groups of New Zealand white rabbits (6 rabbits/group), and the booster immunizations were employed at 2-week interval for 3 times. ELISA assay was used for the quantitative detection of the specific antibody in the sera of rabbits, and the proliferation response of spleen cells was detected by MTT assay. It was found that the target gene Tp92 segment about 2103 bp was obtained, and the DNA sequence of Tp92 gene constructed in pcDNA3.1 (+) vector was consistent with the published nucleotide sequence. The homologies of the nucleotide and putative amino acid sequences of Tp92 gene between T.pallidum subsp. pallidum Nichols and various pathogenic treponeme strains were 95.5%-100%. The analysis of immunocytochemistry and Western blotting showed that Tp92 gene segment constructed in pcDNA3.1(+) vector could express a fusion protein with a calculated molecular mass of 77 kDa in HeLa cells and the expressed protein could react with positive blood serum from syphilis patient. The specific antibody IgG titers were observed and the highest titer was 1∶1024 in rabbits after 3 times with pcDNA3.1(+)-Tp92. The proliferation response of spleen cells were significantly higher than that of rabbits injected with pcDNA3.1(+) ( P <0.05). The successful expression of the eukaryotic expression plasmid of Tp92 gene from T. pallidum was obtained in eukaryotic system and strong responses of humoral and cellular immunity was evoked by DNA vaccine of pcDNA3.1(+)-Tp92 in rabbits thus establishing a solid basis for the future studies in the biological activities and for the development of the syphilis DNA vaccine.
文摘To clone and express the recombinant outer membrane protein Tp0453 of Treponema pallidum and to analyze the immuno-reactivity and immunogenicity of the expressed protein, the immuno-dominant epitope of the Tp0453 was amplified from the complete genome of T.pallidum by PCR, subcloned into expression vector pQE32 to generate the recombinant plasmid pQE32/Tp0453, then expressed in E.coli M15 and analyzed by SDS/PAGE and Western blotting. The fusion protein expressed was purified with Ni-NTA affinity chromatography. Its immuno-reactivity was assayed by indirect ELISA, and the immunogenicity was determined by immunization with this fusion protein in New Zealand rabbits. In the present study, a fusion protein of molecular weight about 32 kDa was obtained. As demonstrated by Western blotting, the recombinant protein could react specifically with positive IgG sera of patients with syphilis, and the antibodies against T.pallidum in human sera were successfully detected by indirect ELISA. Both the sensitivity and specificity of ELISA based on the Tp0453 fusion protein as were 100% (30/30) when detected with control sera. In comparison with the results of IgG ELISA with those of TPPA. It was found that the sensitivity of ELISA was 96.8% and the specificity was 100%. The difference of ELISA and TPPA was not significant, and the concordance of results between ELISA and TPPA was 98.2%. In addition, specific humoral responses could be elicited by immunization with the recombinant fusion protein in New Zealand rabbits with a specific antibody titer of 1∶1280 after 3 successive doses of immunization. These results demonstrate that the expressed recombinant fusion protein shows excellent immuno-competence and provide foundation to develop a quick diagnostic kid applied to detect the presence of T.pallidum infections.
文摘The Gpd gene was amplified from the genomic DNA of Treponema the appropriate site of pcDNA3.1 （ ＋ ） vector. The expression of pcDNA3. I was tested with Western blotting and technology of immunoeytochemisty. New munized with the eukaryotic expression recombinant pcDNA3, 1 （ ＋ ）-Gpd pallidum and cloned into （ ＋ ）-Gpd in Hel,a cells Zealand rabbits were imA fusion protein of C, pd with 4.1 kDa has been effectively expressed in HeLa cells, which were detected bv Western blotting and the immunocytochemistry techniques. The New Zealand rabbits were able to elicit the specific antibody after immunization with the nucleic acid vaccine. The antibody titer could reach as high as 1 ： 1024 after 2 weeks of the third injection; and the splenocytes proliferated evidently due to the Gpd protein stimulation. Both the antibody titer and the splenocytes proliferation were higher substantially than those of controls （ P 〈 0.01 ）. All above data will contribute to an experimental basis of further study of the biological function of Gpd protein as well as DNA vaccine for syphilis.
文摘Objective: To evaluate the clinical application of multiplex PCR inthe detection of Treponema pallidum, Herpes simplex virus (HSV), andHaemophilus ducreyi. Method: Three standard strains were used to set up a multiplexPCR (MPCR) for detecting syphilis, herpes genitalis, and chancroidsimultaneously. Samples from 122 patients with genital ulcer disease(GUD) were subjected to MPCR and the results were compared withthose of dark-field microscopy and TP serology, HSV antigen ELISA,and H. ducreyi culture. Results: In the 122 patients with GUD, MPCR identified 34 casesof T. pallidum infection, 40 cases of HSV infection, and 2 cases of mixedinfection of T. pollidum and herpes. No positive results of H. ducreyiwere found. The sensitivity of MPCR to T. pallidum and herpes was100% and 93.3%, respectively. The sensitivities of dark-field mi-croscopy and TP serology, HSV antigen ELISA, and H. ducreyi cul-ture was 35.3%, 50% and 100%, respectively. Conclusion: MPCR showed a relatively higher sensitivity for T.pallidum as compared with the routine techniques. Although its sensi-tivity for HSV was not as good as that of antigen ELISA, it also yieldeda high detection rate. MPCR can detect more than one pathogen. It issimple, quick, sensitive, and suitable for clinical use or epidemiologicalinvestigation.
基金Focal point financial assistance entry(002A046)Department of Education, Hunan Province Scientific Research Foundation(B2003-085) Department of Public Health, Hunan province.
文摘Objective: To construct the recombinant plasmid containing Glycerophosphodiester phosphodiesterase (Gpd) gene from Treponema pallidum and transfect it into Hela cells to express the encoded outer membrane protein. Methods: The Gpd gene was amplified from the genomic DNA of T.pallidum by polymerase chain reaction (PCR) and inserted into cloning vector pUCm-T. The inserted Gpd gene was subcloned into the appropriate site of pcDNA3.1(+) vector. After identification by sequencing and restrictive enzymes digestion, the recombinant plasmid was transfected into Hela cells using liposomes. The expressed protein was identified by immunocytochemistry and Western blot. Results: The target Gpd gene segment was approximately 1059bp. The DNA sequence of the Gpd gene contained in the pcDNA3.1(+) vector was consistent with the published nucleotide sequence. The homology of the nucleotide and putative amino acid sequences of the Gpd gene between T. pallidum subsp. pallidum Nichols and various pathogenic treponemal strains ranged from 98% to 100%. Immunocytochemistry and Western blot analysis showed that the constructed Gpd-pcDNA3.1(+) vector expressed a fusion protein with a calculated molecular mass of 41KDa in Hela cells and that the expressed protein reacted with the sera from syphilis patients. Conclusion: The successful construction and expression of the eukaryotic expression plasmid of the Gpd gene from T.pallidum provide a promising tool to further study the biological activity of T.pallidum and develop a DNA vaccine for syphilis.
文摘Objective: To obtain recombinant Treponema pallidumsubsp pallidum (TP 17KD) lipoprotein in large quantities byamplification and to further purify antigens for laboratorydiagnosis of syphilis and development of a syphilis vaccine. Method: The Tpp17 lipoprotein gene was amplified fromthe TP(strain Nichols), and then it was recombinated into aplasmid pMAL-2c and cloned within E coli l2-TB1. The hostbacteria containing recombinant plasmids were induced withIPTG. The Tpp 17KD lipoprotein gene was amplified by us-ing PCR and positive clones were screened with double diges-tion and PCR. Recombinant plasmids were transformed intoE. coli and the E coli carrying recombinant plasmids wereinduced. The expression of TP 17KD was detected by sodiumdedecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and immunoblot. Results:Gel staining with Coomassie blue G-250 showedthat the induced E coli carrying recombinant plasmid couldproduce 60KD fusion protein at high levels. Gel scanningshowed that 17KD protein expression in E coli accounted for10% of total cellular protein. The recombinant protein antigenreacted with the sera of syphilis patients. Conclusion: Our study lays a cornerstone for developingnew techniques of laboratory diagnosis for syphilis and newvaccines. Preliminary clinical application showed that thefusion protein could be used for the diagnosis of syphilis.
文摘Objective:To apply a regression equation to calculate the gray zone range for detecting Treponema pallidum antibody(TP-Ab)in blood samples using the ARCHITECT i2000SR fully automated microparticle chemiluminescence analyzer.Methods:A total of 180 blood samples initially screened for syphilis antibodies in our hospital were collected,which were tested using both the Treponema pallidum chemiluminescent microparticle immunoassay(TP-CMIA)on the ARCHITECT i2000SR electrochemical luminescence analyzer and the Treponema pallidum particle agglutination assay(TPPA).The optimal regression equation was established based on the TPPA cut-off index(COI)values and positive predictive probability,which was then used to determine the gray zone range for TP-CMIA.Results:This study analyzed 180 serum samples using a binary logistic regression model to evaluate the predictive value of COI values determined by the TP-CMIA method for syphilis.The Hosmer-Lemeshow test yielded P=0.332,further confirming the model's goodness-of-fit.The COI value was identified as a sensitive predictor of syphilis,with the cubic curve model demonstrating optimal fitting performance(R^(2)=0.925).When the positive predictive probability reached 95%,the corresponding COI value was 5.353,indicating that samples with COI values exceeding 5.353 had a true-positive probability>95%.Consequently,the gray zone range for TP-CMIA COI values was defined as 1.000-5.353,with values below this range classified as a false-positive interval.The model accurately classified 98.7%of syphilis cases and 87.0%of non-syphilis cases,achieving an overall correct classification rate of 97.2%.Conclusion:The gray zone range for COI values of TP-Ab detected by the TP-CMIA method(ARCHITECT i2000SR)was established as 1.000-5.353 using a regression equation,with samples showing COI values≤5.353 carrying a potential false-positive risk.
基金the National Natural Science Foundation of China(22325803,22077052,22277042,22107037,22177041,22207042)the China Postdoctoral Science Foundation(2021M691279)the National Key R&D Program of China(2023YFC2308000).
文摘Treponema is a Gram-negative anaerobic bacterium,among which the pathogenic Treponema can cause various diseases,such as venereal syphilis(Treponema pallidum),yaws(Treponema carateum),and oral diseases(Treponema denticola and Treponema medium).Although different from conventional lipopolysaccharides,the extracellular glycoconjugate of Treponema may still be a potential antigen and provide a candidate for vaccine development.Hence,we completed the first chemical synthesis of Treponema medium ATCC 700293 tetrasaccharide precursor containing L-ornithine(L-Orn)and D-aspartic acid(D-Asp)derivatives.The efficiency of non-reducing end disaccharide formation has been improved by optimizing the assembly of the protecting groups in the donors and acceptors.Our[3+1]glycosylation strategy attempted to reduce the length of the acceptor to increase the nucleophilicity of the hydroxyl group,thereby improving the efficiency of synthesizing the target tetrasaccharide.The L-Orn derivative was introduced at the final stage due to its influence on the glycosylation stereospecificity and efficiency.Therefore,the successful introduction of two amino acid derivatives and the synthesis of a tetrasaccharide precursor with complex functional-group modifications have provided valuable insights for synthesizing other complex bacterial glycans.
文摘BACKGROUND The liver is the most common site of digestive system tumor metastasis,but not all liver metastases can be traced back to the primary lesions.Although it is unusual,syphilis can impact the liver,manifesting as syphilitic hepatitis with inflammatory nodules,which might be misdiagnosed as metastasis.CASE SUMMARY This case report involves a 46-year-old female who developed right upper abdominal pain and intermittent low fever that persisted for more than three months.No definitive diagnosis of a tumor had been made in the past decades,but signs of multiple liver metastases were recognized after a computed tomo-graphy scan without evidence of primary lesions.With positive serological tests for syphilis and a biopsy of the liver nodules,a diagnosis of hepatic syphilis was made and confirmed with follow-up nodule reduction after anti-syphilis therapy.CONCLUSION Clinicians must be aware of the possibility that syphilis can cause hepatic inflam-matory masses,especially when liver metastasis is suspected without evidence of primary lesions.A definitive diagnosis should be established in conjunction with a review of the patient’s medical history for accurate therapeutic intervention.
基金supported by the National Natural Science Foundation of China (Grant No. 30800996)the National Natural Science Foundation of Hunan Province (Grant No. 07ii3028)
文摘The tissue destruction characteristic of syphilis infection may be caused by inflammation due to Treponema pallidum and the ensuing immune responses to the pathogen.T.pallidum membrane proteins are thought to be potent inducers of inflammation during the early stages of infection.However,the actual membrane proteins that induce inflammatory cytokine production are not known,nor are the molecular mechanisms responsible for triggering and sustaining the inflammatory cascades.In the present study,Tp0751 recombinant protein from T.pallidum was found to induce the production of proinflammatory cytokines,including TNF-α,IL-1βand IL-6,in a THP-1 human monocyte cell line.The signal transduction pathways involved in the production of these cytokines were then further investigated.No inhibition of TNF-a,IL-1β,or IL-6 production was observed following treatment with the SAPK/JNK specific inhibitor SP600125 or with an ERK inhibitor PD98059.By contrast,anti-TLR2 mAb,anti-CD14 mAb,and the p38 inhibitor SB203580 significantly inhibited the production of all three cytokines.In addition,pyrrolidine dithiocarbamate (PDTC),a specific inhibitor of NF-κB,profoundly inhibited the production of these cytokines.Tp0751 treatment strongly activated NF-κB,as revealed by Western blotting.However,NF-κB translocation was significantly inhibited by treatment with PDTC.These results indicated that TLR2,CD14,MAPKs/p38,and NF-κB might be implicated in the inflammatory reaction caused by T.pallidum infection.
基金This study was supported by a grant from the Natural Science Foundation of Beijing （No. 7102079）.
文摘Background Tp15, Tp17, Tp45, and Tp47 are outer-membrane proteins found in Treponema pallidum, the etiologic agent of syphilis. These proteins are potent antigens and are potential markers for the serological detection of syphilis. The present study analyzed antibodies to these protein antigens （TP-IgM and TP-IgG） in human serum and investigated the expression of these antibodies during different stages of syphilis. Methods Serum samples were collected from 69 subjects （male 45, female 24） diagnosed with syphilis and analyzed by Western blotting for the expression of IgM and IgG against the four protein antigens. Expression levels of the target antibodies were compared during the same stage of syphilis as well as between different stages of this disease. Results In subjects with primary syphilis, the positive rate of Tp45 IgM was higher than that of other TP-IgM. Tp15 IgM was detected only in subjects with tertiary syphilis. Similarly, the seroprevalence of Tp45 IgG in primary syphilis was higher than for other TP-IgG. No target TP-IgM was detected in subjects with latent syphilis. In subjects with secondary syphilis, the expression level of Tp15 IgG （138.73±20.16） was higher than for other target TP-IgG. In subjects with tertiary syphilis, all target TP-IgG were detected. In subjects with tertiary or latent syphilis, the expression levels of Tp45 IgG （121.33±11.04 and 110.10±40.19, respectively） were higher than those of other target TP-IgG. The expression levels of all Tp-lgM were similar before or after anti-syphilis treatment. In comparison, the expression levels of all TP-IgG decreased compared with the pre-treatment levels, and this decrease was statistically significant （both P 〈0.05） for Tp17 IgG and Tp47 IgG. Conclusions After Treponema pallidum infection, Tp45 IgM appeared first and Tp15 IgM occurred during later stages. The positive rates of all TP-IgG increased with the duration of this disease. Anti-syphilis treatment reduced the expression levels of Tp17 IgG and Tp47 IgG. Larger-scale studies are required to further validate the value of Tp15, Tp17, Tp45, and Tp47 as markers for the early detection of primary and latent syphilis.
基金This research was supported by grants from the National Natural Science Foundation of China （No. 30771930） and the Natural Science Foundation of Jiangsu Province （No. BK2010136）. The authors declare no conflict of interest.Acknowledgement： We thank Dr. FU Kang from Sangon Biotech （Shanghai） Co., Ltd. （Shanghai, China） for his technical support in this study. We also thank Crystal Shen from Mayo Medical School （Rochester, USA） for her detailed advice and help.
文摘Background Treponema pallidum （T. pallidum） subsp, pallidum is the causative agent of syphilis. Analysis of recombinant antigens of T. pallidum led to the identification of potential candidate antigens for vaccine development and syphilis serodiagnosis. Tp0965 was predicted to be a membrane fusion protein and was found to be reactive with infected human sera in previous studies, but the results were controversial. In this research, the antigenicity and immunoreactivity of recombinant protein Tp0965 were assessed. Methods T. pallidum subsp, pallidum （Nichols strain） was propagated and isolated and the genomic DNA was extracted. The Tp0965 gene was amplified by polymerase chain reaction （PCR）. Then the recombinant protein Tp0965 was expressed in Escherichia coli and purified by nickel-nitrilotriacetic acid （Ni-NTA） purification system. The reactivities of protein Tp0965 were examined by immunoblot analysis and indirect enzyme-linked immunosorbent assay. The antisera against protein Tp0965 were obtained by immune rabbits and the immunogenicity of antisera were detected by indirect enzyme-linked immunosorbent assay. Results Recombinant protein Tp0965 was expressed successfully in vitro. Immunoblot assay showed that the recombinant protein Tp0965 could be recognized by human syphilitic sera of all stages. Indirect enzyme-linked immunosorbent assay showed there were only 4 of 74 human syphilitic sera that failed to show reactivity to recombinant antigen Tp0965, and lack of reactivity of Tp0965 to all 28 uninfected sera. A low titer of antiserum against Tp0965 in immune rabbits could be detected after the third time of immunization. Conclusions The recombinant antigen Tp0965 shows excellent sensitivity for the reactivity with sera from syphilitic individuals at all stages. The results also demonstrate a potential application for the serodiagnosis of syphilis.
