Alpha-defensin As A Biomarker For The Diagnosis Of Periprosthetic Joint Infection After Arthroplasty

BackgroundPeriprosthetic joint infection(PJI)after arthroplasty is a catastrophic complication,which is clinically unavoidable and often leads to poor clinical outcomes due to its complexity.The treatment often requires a two-step joint revision,which will bring more economic stress and burden to the patient and the health care system.It is reported that the failure rate has been increasing,which will eventually lead to treatment failure.How to diagnose the PJI in a timely and accurate manner,it is very important to avoid unnecessary 2-stage refurbishment during surgery to avoid false-positive cases and to prevent only one stage of repair in false-negative cases.However,in most patients,the diagnosis of PJI is not easy.The symptoms of PJT are often not specific and clinically significant.The diagnosis of PJI is always clinically challenging.Prevention is the primary and the best way to reduce this catastrophic complication.The Musculoskeletal Infection Society(MSIS)has identified defects in this clinical diagnosis and provides definitions and diagnostic criteria for PJI based on clinical manifestations,laboratory tests and histopathology.But it requires 1-2 major indicators or 4-6 secondary indicators.Although it can be used for clinical,but it is complex and time-consuming.PJI is one of the important factors that affect the success of the surgery.In the first joint replacement,the probability of infection in the hip and shoulder joint is usually not more than 1%,knee joint infection is generally not more than 2%,but occurred in the elbow joint infection rate is usually higher,generally about 9%.For patients with joint revision,the infection rate is much higher than the average patient.This infection has a certain mortality rate,it is reported tha the mortality rate induced by PJI is 1.0%?2.7%.The ideal way to diagnose is finding of the highly sensitive and specific biomarker which is easy to master and understand.At present,clinicians and scientists are committed to the development of effective and simple diagnostic tools for the diagnosis of PJI by detecting biomarkers in the joint cavity fluid.But so far,there is still no biomarkers with clinically accepted sensitivity and accuracy.Alpha-defensins are a part of the innate immune system and are a polypeptide which can anti-viral,bacterial,fungal and other microorganisms.The function of a-defensins is primarily a pathogenic microorganism that can directly neutralize the invasion.a-defensins mainly exists in the neutrophils and other cellsincluding mononuclear neutrophils,lymphocytes.In natural killer cells,it also have a certain expression.In the neutrophils,a-defensins play an important role in oxidative stress-dependent killing of phagocytic pathogens.Moreover,a-defensin has a broad spectrum of anti-microbial effects.It has a killing effect to a variety of Gram-negative and positive bacteria.Its important role in antimicrobials suggests that the expression of alpha-defensin in the articular fluid may be a diagnostic biomarker for PJI.However,in this regard,a-defensin detection in the diagnosis of PJI is still no independent institution to confirm,and the sensitivity and accuracyin different reports are still controversial.At present,there is a large number of in vitro studies on the diagnosis of PJI.However,in vitro studies are still difficult to replace the in vivo study.which requires us to establish an efficient and useful animal model.In order to study the diagnostic value of a-defensin in PJI,we prepared the artificial knee prosthesis replacement model by using rabbits.By injecting different concentrations of Staphylococcus aureus into the joint cavity,the infection status and joint fluid volume were observed at different time points to determine the appropriate amount of bacterial and treatment time.The accuracy and specificity of the a-defensin in the diagnosis were measured using the animal model and compared with the conventional laboratory tests,especially the white blood cell count(WBC)polymorphonuclear percent(PMN%),erythrocyte sedimentation rate(ESR),and C-reactive protein(CRP)were used to investigate the diagnostic value of a-defensin for PJI.As the diagnosis procedure of PJI always lack sensitive or specificity and formation of biofilm of bacteria in joint cavity,the diagnosis of PJI has great difficulties.Alpha-defensin,act as an important bacteriostatic peptide,which is secreted by the sub-leaf neutrophils and other cells into the joint fluid inside,and then integrated into the pathogen cell membrane and quickly kill the pathogen.?-defensin detection may become a new molecular biology means to overcome this difficulty.PART 1Animal model of Periprosthetic Joint Infection afterArthroplasty to Evaluate the Fluid Volume in Joint CavityObjective:The current study on the use of Staphylococcus aureus to construct animal models of PJI has been reported in several cases,but the specific amount of bacteria,the time of infection,the amount of synovial fluid produced,the standard and experimental or clinical application of the model is still unclear.The aim of this study was to establish a model of the PJI after optimization of the key factors in the model,such as the amount of bacteria used and the time of infection.Methods:A total of 64 healthy Chinese white rabbits were used in this experiments.The same researcher used the titanium alloy screw knee arthroplasty in the rabbit left knee joint.Then the animals were randomly divided into control group and experimental group(4 groups,the control group A given saline,experimental group B,C,D were given Staphylococcus aureus liquid 1ml,the concentrations were n.5X 104cfu/ml,0.5×105cfu/ml,0.5×106 cfu/ml).And then through the general score,bacterial culture identification,mortality,infection rate,joint puncture fluid measurement and other methods to verify and determine the infection according to MSIS diagnostic criteria and infection grading score.Results:There were no significant differences in the body weight,age and sex ratio between the four groups.After injection of saline or Staphylococcus aureus,the amount of fluid in the first week was measured by MRI(Group A not detectable;Group B,0.57±0.21;Group C,3.05±0.51;Group D,3.27±0.38).The amount of joint fluid in group C and group D is significantly higher than group B(p<0.001).But there was no significant difference between group C and group D(p = 0.38).The volume of joint effusion was measured in the second week after injection(Group A not detectable;Group B,1.58±0.32;Group C,6.18±0.66,Group D,6.25±0.58).The amount of joint fluid in group C and group D is significantly higher than group B(p<0.001).But there was no significant difference between group C and group D(p =0.82).The measurement of joint fluid by fluid extraction is also conducted in the first week(Group A not detectable;Group B,0.12±0.12ml;Group C 0.25±0.32ml;Group D,0.37±0.15ml).Group C and group D was significantly higher than group B(P<0.001),and there was a marginal difference between group C and group D(p =0.042).The second week after injection,(Group A not detectable;Group B,0.14±0.22ml;Group C,0.76±0.11ml;Group D,0.67±0.11).Group C was significantly higher than group B(p<0.001)and group D was also significantly higher than group B(p<0.001).But there was no significant difference between C and D groups(p=0.15).The success rate of B,C and D infection models was 62.5%,100%and 100%,respectively.There was no infection in the control group.Although the infection rates were 100%in both groups C and D,the death rate in group C was 0%and that in group D was 18.75%.At the same time,the experimental data show that the second week after infection with 0.5 X 105cfu/ml bacterial,the amount of joint fluid is enough to do experiments,and to ensure that most of the experimental animals live well.Conclusion:In this study,we successfully constructed the stable animal model of PJI with injection of 1ml of Staphylococcus aureus with a concentration of 0.5×105cfu/ml in rabbit knee joint cavity,and achieved sufficient joint fluid for experimental purpose of PJI in vivo in the 2 weeks after injection.PART 2The Value of a-defension in the Diagnosis of Periprosthetic Joint Infection after ArthroplastyObjective:To investigate the value of a-defensin in the diagnosis of PJI by using the model of PJI in rabbits underwent arthroplasty.The diagnostic thresholds were determined and compared with other diagnostic indicators.To provide experimental basis for clinical application.Methods:90 rabbits were randomly divided into three groups:group A was blank control group,group B was experimental control group and group C was postoperative infection group.Two weeks after the successful preparation of the model,the blood from the ear vein was used for the detection of leukocyte,percentage of neutrophils,C-reactive protein and erythrocyte sedimentation rate.After the animals were sacrificed by air embolization,the rabbits in group A and B were injected with 1ml of sterile saline,and then washed out to obtain samples,the group C obtain synovial fluid throught direct puncture.The expression of a-defensin in articular fluid was measured by double antibody sandwich enzyme-linked immunosorbent assay(ELISA).The infection was detected by culturing intraperitoneal synovial fluid,intra-articular synovial tissue,inflammatory granulation tissue surrounding prosthesis and prosthesi.ROC curve and Pearson correlation analysis were used to detect cut-off value of a-defensin and determine the specificity and sensitivity.Result:There were 30 animals in each group.According to the results of bacterial culturing,the growth of Staphylococcus aureus was observed group C,but not in group A and B,which proved that the infection was Staphylococcus aureus.The MSIS standard was used to diagnosis PJI.The infection model was established successfully.The number of white blood cells in group C was significantly higher than group A and B(6.59±1.2×109/L in group A,10.15±1.07×109/L in group B,11.76±1.55×109/L in group C,p<0.001),sensitivity and specificity is 96%and 67%,respectively.The percentage of neutrophils was significantly different between the three groups(41.78±10.45%in group A,56.00±11.09%in group B and 69.17±10.26%in group C,p<0.001),and the sensitivity and specificity were 100%And 64%.The ESR was also significantly different between the three groups(Group A,2.94±0.05mm/h,group B,13.43±0.91mm/h,Group C,17.48±2.71mm/h,P<0.001),the sensitivity and specificity was 82%and 67%,respectively.CRP was significantly different between the three groups(group A 1.67±0.15mg/L,group B 1.82±0.15mg/L,group C 2.20±0.27mg/L,p<0.001),diagnostic specificity and sensitivity is 82%and 87%,respectively.The detection results of ?-defensin were significantly higher in group C than in the other two groups(36.56±0.87 ng/?L in group A,37.91±1.02 ng/?L in group B,59.15±5.64 ng/?L in group C,p<0.001).Alpha-defensin in the diagnosis of specificity and sensitivity were 100%and 95%,much higher than other indicators.Conclusion:Alpha-defensin as a diagnostic marker of PJI has a high sensitivity(100%)and specificity(95%)and is an ideal diagnostic standard for peripheral infection.But whether it is affected by other factors,it still need other animal models and clinical trial to validate.