Prospective Study Of BK Virus Infection And BK Virus Associated Nephropathy In Renal-transplant Recipients

Objection:1、To design and built the methods and a kit for clinical test of the BK Virus (BKV) infection in renal-transplant recipients by the real-time quantitative PCR,and to be applied to clinical testing.2、To predict BK virus infection trend and to analyze the the clinical related risk factors of influencing BK virus associated nephropathy (BKVAN) progress by testing BKV DNA loads in renal--transplant recipients.3、To investigate the methods for clinical diagnosis and treatment of BK viremia and BKVAN in renal-transplant recipients.Methods:Part Ⅰ Designing and builting the methods and a kit for testing the BK Virus by the real-time quantitative PCR:1、According to the BK virus genome, BKV-F, BKV-Rand Taqman fluorescent probe BKV-P were designed by us.2、he preparation of the reaction liquid with BKV DNA by PCR.3、To detection reaction.4、To process urine and peripheral blood (PB) samples and extract of the BKV DNA.5、To building BKV quantitative standard product Ⅰ-Ⅳ.6、To estimating the reaction result.7、To test and verify the reaction result.8、 To build the kit of testing the BK VirusPart Ⅱ1、We systematically screened for active BKV infection at preoperative and at0.5,1,3,6,9,12and15months after transplantation in116 renal transplant recipients. The screening tests included urine cytology (by the Papanicolaou method) and BKV DNA PCR (the kit for testing the BK Virus) assay of both urine and plasma, and recording test results carefully.2、Renal biopsy was perfornled if the graft function deteriorated gradually or the loads of BKV replication are very high. Routine histopathological examination and immunohistochemist were performed on renal tissues from partial patients who received the tests of renal biopsy.3、All of the examination data was performed statistically using SPSS software (version16.0SPSS), to assess the incidence and intensity of BKV infection and the morbility of BKVAN in116renal-transplant recipients were followed for at least15months, to predict BK virus infection trend and to analyze the risk factors of influencing BK Virus associated nephropathy (BKVAN) progress.4、According to the existing literature material, intervention treatment in the clinical to the patients who are BK viremia and BKVAN after renal-transplant recipients. The levels about BKV DNA loads and the serum creatine concentrations before and after treatment should be compared with each other at the same time.Results:1-Results showed that the sensitivity low limit and high limit of PCR reaction is1×103copies/ml and1.0×1010copies/ml by testing the BK virus with a kit. There are not cross reaction by using the kit of BK virus among BKV\HBV, EBV、HCMV、JCV and HPVB19. Testing urine and peripheral blood (PB) samples are taken1.5-2.0hours by the real-time quantitative PCR with the kit of BK virus.2、Throughout the follow-up of15months, urinary decoy cells (median8/10HPF,[1-48/10HPF]), BKV viruria (median2.63×105copies/ml,[1.78×103--8.54×109copies/ml]), BKV viremia (median2.70×104copies/ml, [1.95×103-6.31×106copies/ml]), and BKVAN (4patients) occurred in53.46%,24.17%,20.72%和3.45%of renal-transplant recipients, respectively. The positive rate of the Decoy cell and BKV DNA in urine were the highest peak time in the third month after transplantation, and the peak time of the BK viremia was the fifth month post-transplantation throughout the follow-up period.3、The change in BKV DNA level remained constant in blood and urine throughout the follow-up period. But the average amount of BKV DNA (in log10copies/mL) was2.56times higher in urine than blood.4、About4cases of BKVAN were diagnosed,their BKV DNA in the urine and PB were positive. BKV DNA loads level of the BKVAN comparing to the none-BKVAN patients in blood and urine were higher.BKV DNA loads level of the BKVAN in blood were5.07×105copies/ml and4.33×105copies/ml of the median, respectively. BKV DNA loads level of the BKVAN in urine were7.36×107copies/ml and5.73×105copies/ml of the median, respectively. BKV DNA loads level of the BKV viruria was5.53×105copies/ml (P=0.02).5、The clinical data of116cases of renal-transplantation were summarized. Potential variables were analyzed by Logistic regression model multivariate analysis to assess and rank the relative risk of BK virus infection and BKVAN. All other factors analyzed were not associated with BKV infection and BKVAN including the presence of diabetes mellitus, age, gender, living donor graft use, Induction therapy, acute rejection, or pneumonia early after transplant.An increased hazard of BKV infection was associated with BK viremia before transplantation (r=0.457, P=0.000)6、The incidence of the positive BKV DNA loads of116cases of renal-transplantation who received CsA (15.52%) was lower in patients compared to FK506(29.31%). The incidence of BKV infection was lower in patients who received CsA compared to FK506(r=0.232, P=0.012). The incidence of BKV viremia was also lower in patients who received CsA-MMF (r=-0.236, P=0.011) compared to FK506-MZR(r=0.197, P=0.034). An increased hazard of Decoy cell in urine was associated with DGF after transplant (r=0.195, P=0.036).7、According to related literature and guide, the24cases who were BKV viremia including4BKVAN patients were therapied by reducing immunosuppression or replacing of FK506with CsA, without increased acute rejection, allograft dysfunction or graft loss.Conclusion:1、Urine and peripheral blood (PB) were taken from116renal transplant recipients for BKV DNA after transplantation by testing the BK virus with a kit.The real-time fluorescent quantitative PCR assay established in this study was qualitative and quantitative, and has the ability of sensitivity and specificity, low-cost and less false-positive. It is used to screen on BK virus infection and BKVAN after transplantation instead of renal allograft biopsy specimens.2、After the clinical practice, the kit for testing the BK virus is treated as non-invasive diagnosis of laboratory testing tools for practical large-scale clinical work, is applicable to monitor and screen on BK virus infection and evaluate and take precautions against BKVAN after transplantation.3、The peak times of BKV infection was apparently three to nine months after transplantation, suggesting the importance of monitoring urine cytology and BKV DNA loads in post-transplantation patients closely during this period in order to reduce BKVAN after transplantation.4、We research and develop the kit for testing the BK virus,which could be used to predict BKVAN after transplantation when the serum BKV load exceeds105copies/ml or the urine BKV load exceeds107copies/ml.So the two indexes can be as a diagnostic BKVAN progress threshold value or positive index. 5、The recipients who are BK viremia before transplantation would be general clinical independent risk factor, diagnosed BK viremia after transplantation. The recipients who received FK506or FK506+MZR would be independent risk factor of evolving BK viremia and BKVAN after transplantation.Monitoring and screening for BK viremia recipients and donors in preoperative and postoperative is one of the effective measures to prevent and reduce the rate of progression to BK viremia and BKVAN.On this basis, we recommend the regular monitoring of BKV activity in recipients of renal transplants to diagnose BKVAN early, to take an appropriate clinical interventions when necessary6、Monitoring and pre-emptive of immunosuppression reduction or replacing of FK506with CsA were associated with resolution of viremia and showed effective in BKVAN recipients at the early stage without acute rejection or graft loss.