The Application Of Nested PCR And Low Avidity Tests For The Detection Of Early Syphilis Infection

Syphilis is a harmful sexually transmitted disease which has currently been in a critical period of increasingly high incidence rate in China, as is revealed by surveillance; meanwhile, syphilis patients are prone to be infected with AIDS. Early diagnosis and timely treatment, therefore, is of primary importance in syphilis prevention.The chancre of primary syphilis patients contains multiple treponema pallidum (TP), with peculiar antibodies of treponema in their serums. While the syphilis can be detected through the TP in the ulcer secretion with sensitivity and special testing methods, early or late infection of syphilis patients can be differentiated by detecting the avidity of the peculiar TP-IgG antibody in the serum.Based on TP polA gene sequence, this paper tests the ulcer secretion and serum of the primary syphilis detected in 31 patients, and initiates a nested PCR technique. The finding shows that the testing technique is of high uniquity in measuring TP Strains and control strains and can measure the TP secretion concentration degrees of up to one bacterium/ul, done only by increasing the TP strains. Applying the PCR technique in measuring the primary ulcer secretion of the 31 cases mentioned above has found a positive rate of 90.32 %, higher than the result by the dark field (DF) approach. The differentiation stands out (χ~2=8.10,P〈0.005), slightly higher than the TPPA method but no statistical difference has been found. Meanwhile, the use of this technique in detecting the TP-DNA in the serum has also found a positive rate of 12.90%, significantly lower than the results found by other PCR and conventional methods. The finding suggests a possibility associated with low TP content in the serum and demonstrates that serum is not an ideal sample to be used in TP-DNA detection.The parallel tests on the DF,TRUST,TPPA,TP-IgM ELISA,TP-IgG ELISA for the 31 cases mentioned above has found positive rates of 54.84%,74.19%,87.1% 61.29% and 74.19% respectively. Compared with the TPPA method, the DF, TP-IgM positive rates are lower and show a outstanding difference; however, compared the positive rate of TP-IgM and TP-IgG, the difference is insignificant (χ~2=0.66, P=0.42>0.05). This research also attempts to determine whether syphilis is of early infection or late infection by examining the variation of antibody avidity index (AI), through the improved ELISA and Western blot (WB) methods employed in detecting the TP-IgG avidity antibody in the 15 patients diagnosed as primary syphilis infection and the 21 cases determined as latent infection. The ELLSA test based on the urea samples with the denaturant 8M has shown a drop of absorbency on seven cases and a rise on eight cases among the fifteen primary syphilis patients of early infection. The test has also indicated a drop of six cases and a rise of fifteen cases among the 21 patients with latent syphilis. These figures demonstrate that this method is not suitable in TP avidity antibody tests, conforming to reports frequently available in literatures.On the other hand, the WB method used to test the urea samples with denaturant 8M has found that the colour of the four antigen bands of 15, 17, 45 and 47 kDa etc display a clear fade-out, indicating fourteen positive cases among the fifteen primary syphilis infections. When tested with Wilcoxon test, it made a significant difference. However, the colour of the four antigen band has also found remarkably faded out by the conventional WB avidity antibody test of the 21 cases in the contrastive group, the data of which being again tested by the Wilsoxon test. It is, therefore, hard to determine the low avidity antibody among the special antibodies. This result is not conformed to Wonzicova and others who report that the 17 kDa antigen band can be used to differentiate early and late syphilis infection, which calls for further research.In conclusion, the nested PCR method has shown its high sensitivity and uniquity in detecting the TP-DNA in the ulcer secretion of primary syphilis patients and can be used to replace the DF method for early diagnosis of primary syphilis. The use of the improved WB approach to detect the low avidity antibody in the syphilis serum, though found not conformed with the expected result and no report in the research area of low avidity antibody both home and abroad, will provide beneficial experience for further research in this domain.