| Background and Objective: In recent years,synovial fluid α-defensins have been considered as promising biomarkers for the diagnosis of Periprosthetic Joint Infection.The quantitative detection of α-defensins is usually performed by enzyme-linked immunosorbent assay(ElISA).However,it has the disadvantages of multiple detection steps and long duration.α-defensin lateral flow test strips have been developed in foreign countries to qualitatively detect α-defensin,which can quickly perform qualitative detection of synovial fluid α-defensin,and Solve the shortcomings of the cumbersome and long time-consuming process of α-defensins ElISA detection,but it cannot perform quantitative detection,it can not provide more useful clinical information(such as α-defensin threshold,interval likelihood ratio).Therefore,we developed the α-defensin fluorescent test strip for rapid quantitative detection ofα-defensins in synovial fluid.By comparing the quantitative detection of α-defensin fluorescence test strip with that of ElISA,the correlation between the quantitative detection of α-defensin fluorescence test strip and the quantitative detection of ElISA was analyzed.If the α-defensin fluorescent test fluorescent test strip quantitative detection is not inferior to ElISA quantitative detection,it will provide a new auxiliary examination method for clinical diagnosis of Periprosthetic Joint Infection.Research methods and results Part I Development of α-Defensin Fluorescent Test Strips Methods A total of 50 cases of artificial joint revisions were required to be performed in several top-three hospitals in Fujian Province from January 2017 to January 2018.According to the diagnostic criteria of PJI(2013 Revision),the patients were divided into infected and non-infected groups.There were 22 infected cases and 28 non-infectedcases.The preoperative or intraoperative joint fluid was measured by enzyme-linked immunosorbent assay(ElISA).According to the measured α-defensin value,The receiver operating curve(ROC)is drawn and the area under the curve(AUC)was calculated.Tables were calculated for sensitivity(Se),specificity(Sp),positive likelihood ratio(+LR)and negative likelihood ratio(-LR).Joint fluid alpha-defense controls A,B,and high value samples were determined from the measured joint fluid samples.Develop fluorescent test strips,then determine the standard curve ofα-defensin fluorescent test strips and set the fluorescence immunoassay analyzer,At last evaluate the performance of α-defensin fluorescent strips including: minimum detection limit,linear range,accuracy,precision,and stability.Results The median concentration of a defensin in the joint fluid of the infected group was 232.5 ng/ml and was significantly higher than that of the control group(P<0.05),105.7 ng/ml.AUC was 0.955 with a threshold of 179 ng/ml,a sensitivity of 90.9%,a specificity of 96.4%,a LR of 25.25,and a LR of 0.094.The joint fluid α-defensive controls A,B,and high-value samples were determined from the measured joint fluid samples.The α-defensin concentration of A was 49 ng/ml,and the α-defensin concentration of B was 190 ng/ml.The high-value sample α-defensin concentration was422 ng/ml.The correlation coefficient between the α-defensin concentration and the fluorescent signal intensity of the fluorescent test strip is R2 = 0.9194,and the correlation curve equation is plotted with high accuracy.The lowest detection limit ofα-defensin fluorescent strips was 0.8ng/ml.Linearity range: In the concentration range of 0 to 400 ng/ml,the linear regression equation was Y=1.0896 x,R2=0.9564,and the linear relationship was good.The difference between the recovery rates of the quality control products A and B and the average recovery rate was less than 10%,and the proportional system errors were 1.83 %and 1.52%,respectively.Both were less than 1/2total allowable error(TEA)and the accuracy was good.The average intra-assay coefficient of variation(CV)of controls A and B was 5.84%,and the average inter-assay CV was 7.63%,all less than 10%.This indicates that the accuracy of α-defensin assay was good with the fluorescent strip method.Two levels of quality control were testedwith reagents placed at room temperature and 37°C for 14 days.The changes in A mean values were 2%,6%,respectively.The changes in mean B values were 3%,8 %and were all less than 10%,indicating that the stability of the fluorescent test strips was comparable.it is good.Part II Preliminary Application of α-Defensin Fluorescence Test Strip in the Diagnosis of Periprosthetic joint infection Method In the same batch of cases tested with enzyme-linked immunosorbent assay,According to the diagnostic criteria of PJI(2013 revision),22 patients were divided into infection group and 28 non-infection group.The α-defensin fluorescent test strip was used to detect the joint fluid α.-Defensin concentration,drawning the receiver operating curve(ROC),calculating the area under the curve(AUC),calculating the sensitivity(Se),specificity(Sp),and positive likelihood ratio(+LR)according to the diagnostic square table With the negative likelihood ratio(-LR),the interval likelihood ratio(iLR)was further calculated to determine the quantitative detection of the test strip.By comparing the quantitative detection of α-defensin fluorescence test strip with that of ElISA,the correlation between quantitative detection of α-defensin fluorescence test strip and ElISA was analyzed.Results Theα-Defensin fluorescent test strips detected a median concentration of α-defensins in the synovial fluid of the infected group of 202 ng/ml and significantly higher than that of the control group(P<0.05),95.5 ng/ml.AUC was 0.920,the optimal threshold was 167 ng/ml,sensitivity was 81.8%,specificity was 89.29 %,+LR 7.64,-LR 0.20).Interval likelihood ratio calculations show that α-defensins levels below 100ng/ml significantly reduce the post-test probability of PJI(iLR 0.12),and α-defensins levels higher than 200 ng/ml resulted in a significant increase in post-test probability(iLR11.36).α-defensin fluorescent test strip and ElISA detection correlation coefficient R2 = 0.902,which has a high correlation.There was no significant difference in the area under the α-defensin fluorescence test strip and the ElISA curve.The difference between the two detection rates was not statistically significant(P>0.05).Conclusion Our results show that the α-defensin fluorescent test strip has the advantages of rapid detection,simple method,and high specificity and sensitivity.It can be used for rapid and quantitative detection of α-defensins in joint fluids in the clinic.It has clinical application value and can be used clinically. |
