Clinical Application Of TP-IgM Antibody Detection By Capture ELISA In Diagnosis And Following-up In The Different Stages Of Syphilis

Syphilis is a kind of sexually transmitted diseases caused (STD) by Treponemal pallidum, which can spread by sexual behavior, blood or placenta. The incidence of infectious syphilis has increased steadily in recent years, which has been a relevant public health issue. The early finding and prompt therapy of infected individuals are critical for successful control of epidemic syphilis outbreaks. Treponemal palladium can invade the tissues and organs all over the body, causing multiple organ diseases, dysfunction to human body and even death. So it is a serious threat to human health. Because syphilis has strength infectiosity and complex course, further it is often asymptomatic in the early and secondary syphilis, it is often neglected. This is not only brought great harm to patient's body, but also speed up the spread of syphilis.Serological testing for syphilis is an important tool in the prompt and accurate diagnosis of all stages of the disease. At present, there are two kind of syphilitic Serological detection in laboratory:One is non-treponemal tests, such as the rapid plasma regain test (RPR), toluidine red unheated serum test (TRUST), etc. This kind of assay detects antilipoid antibody in sera, which often be used as screening test for syphilis. It is often used for diagnosis of relapse and reinfection, too. As these tests can be affected by many factor and often appear biological false-positive and false-negative results, so it is suitable only for secondary syphilis, but not for primary and tertiary syphilis. In addition, it can be used for monitoring the effect of treatments, when it is used for this, seroresistant is often be found, so it's difficult for the doctor to determine the end point of treatment. The other is treponemal tests, such as the Treponema pallidum hemagglutination assay (TPHA), Treponema pallidum agglutination test (TPPA), fluorescent treponemal antibody absorption test (FTA-ABS), etc. This kind of assay detects anti-Treponema pallidum antibody. It has high sensitivity and specificity in diagnosis of syphilis, so it is often used as confirmatory test in clinical laboratory. But these tests detect Treponema pallidum IgG antibodies and IgM antibodies, while IgG antibody persistence after infectation and the serum is positive all the time. So it can't be used for monitoring the effect of treatments. Therefore, it is urgent to find a sensitive, specific method for early detection of syphilis, so that provide the exact experimental evidence for the early diagnosis, early treatment and efficacy monitor after syphilis treatment.At the early stage of syphilis infection, specific IgM antibody to Treponemal pallidum (TP-IgM) develops as the first sign of the humoral immune response, which can be detected about two weeks after infection. The TP-IgM antibody was considered a reliable marker of active syphilis infection. If The TP-IgM antibody appears again after curing, it may be re-infection or recrudescence of syphilis. Compared to IgG, IgM antibody has a larger molecular weight and is not able to transfer across the blood-brain barrier or placenta. Demonstrating IgM antibody in cerebral spinal fluid (CSF) or blood of neonates is of particular importance in serological diagnosing neurosyphilis and congenital syphilis. In all, the significance of TP-IgM in syphilis is early detection, treatment evaluation, surveillance, and detection of congenital or neurosyphilis syphilis. In an attempt to further explore and better understand the clinical value of specific IgM in treponemal infection, and evaluate the follow-up significance of TP-IgM after treatment, we designed and performed this study.Over the years, with the rapid development of molecular biology, immunology and genetic engineering technology, recombinant antigens has been successfully developed and gradually used in research of many kinds of serological diagnosis methods and development of relating diagnosis kit. But it is rare in the study about TP-IgM antibody detection in early diagnosis and following-up in our country. In this study, a large number of clinical samples are detected by Capture-ELISA.The result proves that this assay has higher sensitivity and specificity; it is superior to RPR in early diagnosis of syphilis and clinical monitoring. So it is a good kind of therapeutic targets and lays the theoretical and experimental basis for improving the prevention and treatment of syphilis.Materials and Methods1.Materials Patient:128 cases of syphilis were analyzed from the infection of department at the Shenyang City sixth people hospital, through a period from March 2008 to March 2010. Diagnosis was made by disease history, clinical manifestation, RPR test and TPPA test.Treponema Pallidum Particle Agglutination (TPPA) kit was purchased from Fuji Limited company in Japan;Rapid Plasma Regain (RPR) Test kit was purchased from shanghai Rongsheng Biotech Limited Company;TP-IgM ELISA kit was kindly provided by Beijing Hotgen Biological Technology;High speed centrifuge (Sigma, USA);Enzyme guiding meter (BIO-RAD Model 680);Microplate Washing (BIO-RAD Model 1575 Immun Wash);Electrothermal homoeothermic incubation (DRP-9082).2.Methods(1)Collection of Serum Samples:2ml of blood were drawn and sera were isolated.(2)Serum samples of 128 patients with syphilis who is untreated were tested by Rapid Plasma Regain Test (RPR) and T.pallidum particle agglutination (TPPA) for screening and confirmatory syphilis, respectively.(3)Serum samples of 128 patients including various stages of untreated syphilis and serum samples of 10 patients with Rheumatoid Factor positive were tested by Cap-ELISA for detection of IgM to Treponema pallidum, Results were compared with those of Treponema pallidum particle agglutination (TPPA) assay.(4)The patients with TP-IgM positive were followed up by RPR test and Tp-IgM-Cap-EL1SA at 1 months,3 months,6 months and 12 months after treatment.Resultsl.The sensitivity of IgM antibody was 70.8%(14/18) in primary syphilis,100% (24/24) in secondary syphilis, and there was no significant difference between these values. The sensitivity of IgM antibody in diagnosing latent syphilis was only 30.2% (26/86),much lower than the detection rate in early syphilis.The difference was of high statistical value (x2=12.42, P<0.05). Capture ELISA TP-IgM results were not found in the 10 serum samples containing rheumatoid factor.2. The TP-IgM ELISA showed similar sensitivity comparable to that of TPPA at primary syphilis and secondary syphilis.But in the latent syphilis, the sensitivity of the capture TP-IgM ELISA test was lower noticeably than that of the TPPA test. The difference was of high statistical value (x2=120.56, P＜0.05).3. After treat 12 month, TP-IgM became negative in 100%,91.6%, and 50% of patients in primary syphilis, secondary syphilis and latent syphilis respectively. While the RPR test became negative in 92.8%,83.3%,46.1% of patients after 12 months. TP-IgM turns to negative in 12 months after treatment for nearly all patients with early syphilis. In lantern syphilis, there are only 50% patients to become negative. Tp-IgM-Cap-ELISA was found to be negative earlier than that of RPR test in all cases. No seroresistant was shown in TP-IgM assay.Conclusion1. The Cap-ELISA for detection of TP-IgM is easy to use and has high sensitivity and specificity in diagnosis of syphilis, so it is suggested that Cap-ELISA is an alternative laboratory diagnosis for early syphilis.2. When to be used for monitoring treatment efficacy, Tp-IgM was found to be negative earlier than that of RPR test in most cases. No seroresistant was shown in Tp-IgM assay. TP-IgM-Cap-ELISA may be served as a useful tool to follow up syphilis patients after treatment.