Evaluation Of Therapeutic Efficacy And Disease Activity In Early Syphilis Based On Real-time Quantitative PCR

Background:Currently,the assessment of the syphilis activity and treatment are extremely difficult and lack of efficacy evaluation criteria.Therefore,clinicians usually,used serum nontreponemal test antibody titers and its four-fold decline in titers as a judgment index for syphilis activity and treatment efficacy evaluation.However,the dynamic change of the nontreponemal test antibody titers during the Treponema pallidum(T.pallidum)infection course is still unclear.In this study,we used a rabbit model challenged with different T.pallidum strains to examine dynamic change in the Rapid Plasma Reagin(RPR)titer,lesions and T.pallidum mRNA and DNA level during the antibiotic treatment and infection process,and further the infected organs extractions into rabbits’ testis to observe the rabbit infective test(RIT)positive rate as well as the effectiveness of antibiotic treatment,so as to explore the application value of real-time quantitative PCR(qPCR)in syphilis activity and therapeutic efficacy evaluation.Methods:Ten seronegative New Zealand white male rabbits were inoculated intradermally at ten sites on their clipped backs with T.pallidum Nichols and Amoy strain,respectively.Then,the rabbits were further divided into two subgroups:a Benzathine penicillin G(BPG)treatment subgroup and non-BPG control subgroup.Serum samples,lesion biopsies,and different organs were collected at scheduled time intervals for RPR titers,DNA,and mRNA detection,and RIT of different organs.Results:The results indicated that the lesion diameters were significantly greater in the non-BPG control subgroups than in the BPG treatment subgroups challenged with the same strain(p<0.05);however,the serum RPR titers did not significantly differ between BPG-treatment subgroups and non-BPG control subgroups(p>0.05).The RPR titers in the Amoy strain-challenged rabbits were generally higher than those in the Nichols strain-challenged rabbits.Interestingly,a four-fold decrease in RPR titer was observed not only in the BPG-treated rabbits,but also in the non-BPG-treated rabbits.The rate of a four-fold decrease in RPR titers would be depending on the different baseline titer.After BPG treatment,the lesion DNA level was significantly decreased compared with that in the non-BPG-treated rabbits(p<0.05),consistent with disappearance of the lesions.We also found lesion mRNA level was significantly decreased compared with that in the non-BPG-treated rabbits(p<0.05),and after a week of treatment,lesion mRNA was almost undetectable.In addition,the positive rate of RIT infected with the mixed testis and lymph nodes extractions from non-BPG-treated rabbits was consistent with its T.pallidum mRNA positive rate(5/6,83%),and the positive rate of T.pallidum nucleotides(DNA and mRNA)in testis(11/12,92%)was higher than lymph notes(7/12,58%).In addition,T.pallidum mRNA had a higher sensitivity(89.9%)for activity than four-fold decrease of RPR titer(30%)and similar to that of RIT(66.7%),and specificity had no difference among them.Conclusions:In rabbits with early syphilis,a four-fold decrease in the RPR titer should not be used to assess therapeutic efficacy.Instead,the T.pallidum mRNA expression level and variation of DNA load in lesions may be used for evaluating syphilis activity and therapeutic efficacy.In addition,detection of T.pallidum mRNA in testis and lymph nodes could be a potentially useful measure for assessment of the syphilis activity instead of RIT.