BACKGROUNDTp15, 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.

METHODSSerum 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.

RESULTSIn 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-IgM 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.

CONCLUSIONSAfter 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.

Adolescent ; Adult ; Aged ; Antibodies, Bacterial ; blood ; Female ; Humans ; Immunoglobulin G ; blood ; Immunoglobulin M ; blood ; Male ; Middle Aged ; Syphilis ; diagnosis ; Treponema pallidum ; immunology

Syphilis, a chronic bacterial infection caused by the spirochete Treponema pallidum subsp. pallidum, remains a public health concern worldwide. Syphilis control is largely dependent upon early identification and prompt treatment. The diagnosis of syphilis is mainly based on laboratory tests, especially serology and dark-field microscopy. In recent years, recombinant Treponema pallidum antigen-based rapid tests, polymerase chain reaction, and immunoglobulin M antibody detection have also shown certain sensitivities and specificities for syphilitic patients at different stages.
Antigens, Bacterial ; blood ; Humans ; Immunoglobulin M ; blood ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; Syphilis ; blood ; diagnosis ; Syphilis Serodiagnosis ; Treponema pallidum ; immunology ; isolation & purification

BACKGROUNDTreponema 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.

METHODST. 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.

RESULTSRecombinant 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.

CONCLUSIONSThe 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.

Animals ; Antigens, Bacterial ; genetics ; immunology ; Bacterial Proteins ; genetics ; immunology ; Enzyme-Linked Immunosorbent Assay ; Humans ; Membrane Proteins ; genetics ; immunology ; Polymerase Chain Reaction ; Rabbits ; Syphilis ; immunology ; microbiology ; Treponema pallidum ; immunology ; metabolism

BACKGROUNDRecent studies have reported overall increasing rates of syphilis with a high rate of human immunodeficiency virus (HIV) co-infection. However, there is little information about factors influencing syphilis treatment failure and/or re-infection in HIV co-infected patients. We conducted a study to evaluate factors associated with syphilis treatment failure/re-infection in HIV co-infected patients.

METHODSWe reviewed 3542 medical records of HIV-infected patients from January 2005 to December 2007 followed up at HIV Clinic in New York City. Patients were categorized by rapid plasma regain titer (RPR) into success/serofast (4-fold decrease in RPR by 12 months after treatment, RPR conversion to nonreactive, persistently stable reactive RPR with no 4-fold increase), and failure/re-infection (failure to decrease 4 folds in RPR by 12 months after treatment, 4-fold increase in RPR from baseline).

RESULTSAmong a total of 156 patients who met the eligibility criteria, 122 (78.2%) were under success/serofast category, and 34 (21.8%) were under failure/re-infection category. HIV viral load, CD4 cell count, and use of highly active antiretroviral therapy (HAART) were not associated with syphilis treatment failure/re-infection. However, early syphilis stage (OR: 11.036, 95%CI: 2.499 - 48.740, P = 0.002) and high (> 1:64) RPR titers (OR: 715.921, 95%CI: 422.175 - 23 113.396, P < 0.001) were significantly associated.

CONCLUSIONSNo correlations were seen with depressed immune states with syphilis treatment failure and/or re-infection. However, association with early stage syphilis suggests that risky psychological sexual behaviors may be the most important leading factor, emphasizing needs for safe sex education.

Adult ; Antiretroviral Therapy, Highly Active ; methods ; CD4 Lymphocyte Count ; Coinfection ; drug therapy ; immunology ; Female ; HIV Infections ; drug therapy ; immunology ; Humans ; Immunosuppression ; Male ; Middle Aged ; Retrospective Studies ; Syphilis ; drug therapy ; immunology ; Treatment Failure

OBJECTIVETo study the incidence and risk factors of HIV and syphilis seroconversion among men who have sex with men (MSM) in Beijing.

METHODSA total of 550 MSM were recruited on the basis of community and followed up after 6 and 12 months in Beijing. Each subject was investigated by only one investigator at one time to collect information on demographics and behaviors. Blood samples were collected to test HIV and syphilis seroconversion. ELISA was used for screening test, west blotting (WB) and Particle agglutination were used for confirmatory test.

RESULTSA total of 550 MSM investigated, among which 4.5% (25/550) were HIV-positive and 29.3% (161/550) were syphilis-positive. For 525 HIV-negative MSM, 87.0% (457/525) retained during the 12-month investigation. Seroincidence for HIV and syphilis were 3.37/100 person-years (95%CI = 1.66 - 5.08) and 9.32/100 person-years (95%CI = 5.87 - 12.77) respectively. HIV seroconversions for those who performed and did not perform rectal douching after homosexual anal intercourse in the past 3 months were 7.11/100 and 0.76/100 person-years respectively. Multivariate Cox regression analysis revealed that rectal douching after homosexual anal intercourse in the past 3 months (HR = 9.23, 95%CI = 2.08 - 40.88) was significantly associated with HIV seroconversion. Syphilis seroconversions for those who met male sex partners in parks, public washrooms or bathhouses in the past 3 months were 41.77/100 and 7.97/100 person-years respectively. Syphilis seroconversions for those who performed and did not perform rectal douching after homosexual anal intercourse in the past 3 months were 16.17/100 and 4.92/100 person-years respectively. In the past 3 months, meeting male sex partners in parks, public washrooms or bathhouses (HR = 4.67, 95%CI = 1.77 - 12.34) and performing rectal douching after homosexual anal intercourse (HR = 3.09, 95%CI = 1.40 - 6.83) were significantly associated with syphilis seroconversion.

CONCLUSIONThe seroconversions of HIV and syphilis during the follow-up visits in this MSM cohort study in Beijing were very serious, and that the associated factors for seroconversions were rectal douching after homosexual anal intercourse and meeting male sex partners in parks, public washrooms or bathhouses.

Adolescent ; Adult ; Antibodies, Bacterial ; blood ; China ; epidemiology ; HIV ; immunology ; HIV Antibodies ; blood ; HIV Infections ; blood ; epidemiology ; HIV Seropositivity ; blood ; epidemiology ; Homosexuality, Male ; Humans ; Incidence ; Male ; Risk Factors ; Sexual Behavior ; Syphilis ; blood ; epidemiology ; Treponema pallidum ; immunology ; Young Adult

OBJECTIVETo summarize the clinical features and analyze the serologic test results of latent syphilis.

METHODSThe clinical data of 601 patients with latent syphilis who were treated in the sexually transmitted disease centre of Peking Union Medical College Hospital between January 2001 and November 2007 were retrospectively analyzed.

RESULTSOf the 601 cases of latent syphilis, there were 174 cases of early latent syphilis(EL), 170 cases of late latent syphilis(LL), and 257 cases of unknown latent syphilis. Male to female ratio was 0.74:1256 males and 345 females, respectively). Patients aged 20-39 years accounted for the largest proportion. Non-marital sexual intercourse was the main route of infection. Forty-six patients (7.65%) were co-infected with other sexually transmitted diseases. A total of 251 cases of latent syphilis (41.76%) were confirmed when the patients were receiving tests for other sexually transmitted diseases or suspected sexually transmitted diseases. Of the 601 patients with EL, LL and unknown latency, the proportion of serum rapid plasma reagin(RPR) titers higher than or equal to 8 were 72.99% (127/174), 52.94% (90/170), and 60.31%(155/257), respectively. Compared with the early syphilis, serological negative conversion rate was significantly lower after treatment for l2 months in the early latent syphilis patients (P=0.044).

CONCLUSIONEducation and awareness raising on syphilis should be strengthened to lower the prevalence of latent syphilis.

Adult ; Female ; Humans ; Male ; Middle Aged ; Retrospective Studies ; Syphilis, Latent ; blood ; diagnosis ; immunology ; therapy ; Young Adult

Using recombinant TpNs proteins of Treponema pallidum as antigens, ELISAs are proved to be of higher sensitivity and specificity. However, they can be further increased by using multiple TpNs antigens. According to the epitope analysis, we firstly used linking primers PCRs to obtain an artificial fusion gene segment tpE17-47 containing epitopes of both TpN17 and TpN47. Subsequently, we conducted the prokaryotic expression systems of entire tpN17 and tpN47 genes and tpE17-47 fusion gene. SDS-PAGE analysis and BioRad Gel Image Analysis System showed that the recombinant proteins rTpN17, rTpN47 and rTpE17-47 expressed stably, with 36%, 20% and 28% yields of total bacterial protein, respectively. After purified by Ni-NTA affinity chromatography, all the three recombinant proteins could be recognized by T. pallidum antibody positive sera from syphilis patients. The positive rate of rTpE17-47-ELISA for detecting serum specimens in clinically 630 cases with syphilis was 98.6%. This rate was slightly higher than that by Treponema pallidum particle agglutination (TPPA) (97.9%) (P > 0.05), but significantly higher than those by rTpN17-ELISA (83.8%), rTpN47-ELISA (83.3%) and rapid plasma reagin (RPR) (72.1%) (P < 0.01). Furthermore, both ELISAs and TPPA for detecting the serum specimens in 25 cases with SLE, 36 cases with RA and 250 healthy cases were all negative. RPR showed positive in 1 case with SLE, 2 cases with RA and 2 healthy cases. This could be a novel serological screening or diagnostic method of syphilis with advantages of quickness, convenience, safety, sensitivity and specificity.
Amino Acid Sequence ; Antigens, Bacterial ; biosynthesis ; genetics ; Base Sequence ; Enzyme-Linked Immunosorbent Assay ; Humans ; Immunodominant Epitopes ; immunology ; Molecular Sequence Data ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Sensitivity and Specificity ; Syphilis ; diagnosis ; microbiology ; Syphilis Serodiagnosis ; methods ; Treponema pallidum ; immunology

OBJECTIVETo clone tpn17 and tpn47 genes of Treponema pallidum and then construct their prokaryotic expression systems,to establish ELISAs based on rTpN17 and rTpN47 as antigens and to evaluate the sensitivity and specificity of the ELISAs for detection of serological diagnosis of syphilis.

METHODSThe whole length of tpn17 and tpn47 genes was amplified by PCR and then their prokaryotic expression systems were constructed. SDS-PAGE was used to measure the expression of the target recombinant proteins rTpN17 and rTpN47. Ni-NTA affinity chromatography was applied to extract rTpN17 and rTpN47, while Western blot was performed to determine the specific immunoreactivity of rTpN17 and rTpN47. By using rTpN17 and rTpN47 as the coated antigen, respectively, ELISAs (rTpN17-ELISA and rTpN47-ELISA) were established to detect serum samples from 200 healthy individuals, 25 RA patients, 17 SLE patients and 211 syphilis patients. The detection effects of the ELISAs were compared to those of TRUST and TPHA.

RESULTThe sequence similarity of the cloned tpn17 and tpn47 genes was 100 % compared with the corresponding sequences in GenBank. The expression outputs of rTpN17 and rTpN47 were approximately 37.2 % and 26.8 % of the total bacterial proteins, respectively. Both the extracted rTpN17 and rTpN47 could take place remarkable conjugation reactions to the sera with positive antibody against Treponema pallidum.The positive detection rate of TPHA (99.1%) was the highest (P<0.001). The positive detection rates of rTpN17-ELISA (85.3 %) and rTpN47-ELISA (84.3 %) were similar (P>0.05). The positive detection rates of TRUST (72.5 %) was lower than that of rTpN17-ELISA (P=0.001) but similar to that of rTpN47-ELISA (P=0.014). The detection results of all the serum samples from healthy individuals, RA patients and SLE patients were negative, whereas 7.1 % (3/42) of the samples from RA or SLE patients were positive.

CONCLUSIONrTpN17 and rTpN47 are still maintaining their original immunoreactivity. The ELISAs using rTpN17 or rTpN47 as the antigen are rapid, simple and convenient, higher sensitivity and specificity methods for serological screening and detection of syphilis.

Antibodies, Bacterial ; Antigens, Bacterial ; Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; methods ; Female ; Humans ; Male ; Syphilis ; diagnosis ; Syphilis Serodiagnosis ; Treponema pallidum ; chemistry ; immunology ; isolation & purification

OBJECTIVETo investigate the incidence of seroresistance of syphilis and analyze its relevant factors.

METHODSThe clinical data of 131 patients with syphilis were retrospectively analyzed. The incidence of seroresistance was investigated and the correlation between seroresistance and factors including age, gender, original titer, disease course, and medications were analyzed.

RESULTSThe incidence of seroresistance was not significantly different among patients with different ages and genders, but was significantly different among patients with different disease courses, antibody titers, and medications. Patients with a baseline serum rapid plasma reagin (RPR) titer of greater than 1: 8, a latent course, or a macrolide therapy history had higher incidences of seroresistance (i. e., 61%, 45.6%, and 72.7% respectively) than those who had a lower RPR titer, a primary course, or a benzathine penicillin therapy history.

CONCLUSIONSyphilis patients with a high baseline RPR titer, a latent course, and a macrolides therapy are prone to be seroresistant.

Adolescent ; Adult ; Aged ; Female ; Humans ; Macrolides ; therapeutic use ; Male ; Middle Aged ; Reagins ; blood ; Syphilis ; blood ; diagnosis ; drug therapy ; immunology ; Syphilis Serodiagnosis ; methods ; Young Adult

OBJECTIVETo assess the sensitivity, specificity, and feasibility of 4 recombinant Treponema pallidum antigen-based rapid tests in the diagnosis of syphilis.

METHODSA 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. pallidum 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.

RESULTSThe 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).

CONCLUSIONSThe 4 rapid tests show good performance and characteristics in the diagnosis of syphilis. Furthermore, they are more sensitive for serum specimens than whole blood.

Antigens, Bacterial ; immunology ; Humans ; Quality Control ; Recombinant Proteins ; immunology ; Sensitivity and Specificity ; Syphilis ; diagnosis ; Treponema pallidum ; immunology