Establishment And Preliminary Clinical Application In Neurosyphilis Of Luciferase Immunosorbent Assay Based On Tp Recombinant Protein

Objective1.To analyze the risk factors associated with the development of neurosyphilis by reviewing HIV-negative syphilis cases and evaluating the feasibility of using serological tests as indications for lumbar puncture.2.To construct a luciferase immunosorbent assay with treponema pallidum recombinant proteins Tp0435 and Tp0574 as specific antibodies,and to evaluate the significance of this method in the diagnosis of neurosyphilis,so as to provide certain experimental basis for the exploration of new methods for the diagnosis of neurosyphilis.Methods1.Retrospective analysis was performed on the clinical data and laboratory results of 145 hospitalized HIV-negative syphilis patients who received CSF examination.According to their clinical manifestations and CSF examination results,53 patients were confirmed as neurosyphilis patients and 92 as syphilis patients.The risk factors of neurosyphilis were analyzed by comparing the general data,clinical features,Tp serological test and blood biochemical test of the two groups.To evaluate the predictive value of serum TRUST in neurosyphilis.2.The gene sequences of Tp0435 and Tp0574 were obtained from GenBank,and the whole gene fragment was expanded by PCR with the standard strain genome DNA of TpNichols as the template.The eukaryotic expression vectors of pNLF1-N-Tp0435 and pNLF1-N-Tp0574 were constructed and transfected into HeLa cells after enzyme digestion and sequencing identification,and the cell lysis solution was extracted.Protein G was used to coat 96-well plates,and after incubation,samples to be tested,cell lysates and luciferase substrates were added successively to establish Tp0435-LISA and Tp0574-LISA,and specific antibody levels were determined by detecting light units.The serum and cerebrospinal fluid of neurosyphilis patients and syphilis patients were tested respectively to preliminarily evaluate the application value of Tp0435-LISA and Tp0574-LISA in the diagnosis of neurosyphilis.Results1.The results of single factor analysis showed that gender,age,serum TRUST titration,neurological symptoms,CSF chloride,creatine kinase,creatine kinase isoenzyme,alkaline phosphatase,total bilirubin,urea nitrogen,creatinine,uric acid were correlated with the occurrence of neurosyphilis.Multivariate Logistic regression analysis showed that gender and serum TRUST were risk factors for neurosyphilis.The serum TRUST titer increased and the non-neurosyphilis patients decreased.ROC curve analysis showed that the optimal threshold of serum TRUST was 1:8,which could screen 77.4% confirmed cases of neurosyphilis.2.The 483 bp and 1318 bp target fragments were obtained by PCR amplification.The eukaryotic expression systems of pNLF1-N-Tp0435 and pNLF1-N-Tp0574 confirmed that the inserted genes were Tp0435 and Tp0574 after double enzyme digestion and sequencing identification.3.The Tp0435-LISA was established to detect the serum and cerebrospinal fluid of 20 and 43 cases of neurosyphilis,with sensitivity of 80% and 100%,specificity of81.4% and 97.7%,respectively.The Tp0574-lisa was established to detect the serum and cerebrospinal fluid of 20 and 43 cases of neurosyphilis,with sensitivity of 90%and 100%,and specificity of 55.8% and 69.8%,respectively.4.Tp specific antibody test was carried out on 63 syphilis CSF samples.The sensitivity of CSF-Tp0435-LISA was 100%,the specificity was 97.7%,the negative likelihood ratio was 0,and the positive likelihood ratio was 43.48%.The sensitivity of CSF-Tp0574-LISA was 100%,the specificity was 69.8%,the negative likelihood ratio was 0,and the positive likelihood ratio was 3.31.The sensitivity,specificity,negative likelihood ratio and positive likelihood ratio of CSF-TPPA were 95%,58.1%,8.61 and 2.27 respectively.The sensitivity,specificity,negative likelihood ratio and positive likelihood ratio of CSF-FTA-ABS were 100%,65.1%,0 and 2.87 respectively.5.The results of consistency test showed that the Kappa value of CSF-VDRL and CSF-Tp0435-LISA was 0.964,indicating that the two had very high consistency.The Kappa value of CSF-VDRL and CSF-Tp0574-LISA was 0.621,indicating a high consistency between them.Conclusion1.Gender and serum TRUST titer can be used as auxiliary indicators to predict neurosyphilis.Serum TRUST can screen neurosyphilis,and patients with titer ≥1:8are more likely to develop neurosyphilis.This critical value can be used as a serological indicator for cerebrospinal fluid examination in HIV-negative syphilis patients,and lumbar puncture can be reduced in syphilis patients with serum TRUST<1:8.2.To construct eukaryotic expression systems pNLF1-N-Tp0435 and pNLF1-N-Tp0574,the established Tp0435-LISA and Tp0574-LISA have high sensitivity and high specificity for the diagnosis of neurosyphilis.The detection of specific antibody levels in serum Tp0435-LISA and Tp0574-LISA is of great significance in the prediction of neurosyphilis.3.CSF-Tp0435-LISA is a new diagnostic method with great potential.CSF-Tp0574-LISA,CSF-TPPA,CSF-FTA-ABS have high sensitivity and high negative predictive value,which can be used for the exclusion diagnosis of neurosyphilis.