Pre-emptive Prophylaxis Of HCMV Pneumonia In Renal Transplant Recipients By Dynamic Monitoring HCMV-DNA Load

BackgroundHuman cytomegalovirus(HCMV) is one of the most common pathogens in the early stage of renal transplant recipients.Parts of these patients may develop into HCMV disease.Among them,HCMV pneumonia is difficult to be cured.It develops rapidly and cost mostly.The death rate of severe pneumonia caused by HCMV infection exceeds 65%. It is the most common death reason in the early stage after renal transplantation.Among the transplant recipients,initial infection with,or reactivation of,HCMV can cause direct effects,including an acute viral syndrome with fever and other constitutional symptoms and various endorgan syndromes such as pneumonitis,enterocolitis, nephritis,and hepatitis.In addition to these primary effects,HCMV infection may cause indirect effects,including allograft injury and acute and chronic rejection,increased risk of cardiac complications, secondary bacterial or fungus infection,malignant tumor,diabetes mellitus,and post transplant lymphoproliferative disorder(PTLD). Ultimately,these effects may have great impact on kidney and patient survival rate.In recent years,the infection rate of HCMV in the early stage after renal transplantation show rising trend.Many transplantation centers adopt different prevention strategy to cut down the infection rate of HCMV.Now,the main prevention strategy includes universal prophylaxis strategy,targeted prophylaxis strategy and pre-empty prophylaxis strategy.The universal prophylaxis strategy is to perform antivirus therapy during a special stage(usually 3 months right after transplantation) in all recipients.The targeted prophylaxis strategy is to perform antivirus therapy with ganciclovir only in recipients with high risk. The pre-empty prophylaxis strategy is to select the recipient who is easy to develop into HCMV disease by routine monitoring usually with PCR or antigenaemia analysis,and treat them with ganciclovir. Having read lots of papers,I think the last one is fit for China and show better clinical practical perspective.Nowadays,the main problome is that the prevention strategy haven't attracted doctors and recipients mostly.We don't have a union opinion on the technique of monitoring HCMV.Most of transplantation centers haven't performed a routine monitoring on HCMV.The stage of monitoring the HCMV-DNA load after transplantation in blood preparation and urine aliquot haven't been determined.The standard of HCMV-DNA load to perform pre-empty therapy hasn't been determined.We are not sure about how to use the drug to perform pre-empty prophylaxis strategy.On the aspect of HCMV prophylaxis, the difference between China and the international is obviously.So we have to study hard to learn more about the HCMV prevention strategy. This clinical research closely connected with clinic and it can guide the clinic.In this study,first of all,we test HCMV-DNA copies in blood preparation and urine aliquot basing on TaqMan technology and investigate its practical value.Having analyzed the data on dynamic monitoring HCMV-DNA load with RT-PCR at our transplantation center, we think that RT-PCR is in good uniformity with the test technique of pp65 antigenaemia.Its sensitivity surpasses the test technique of pp65 antigenaemia and HCMV-DNA appears early than pp65 antigenaemia.So,real-time fluorescent quantitative PCR is fit for being used in pre-empty prophylaxis strategy as a monitor technique. We analyzed retrospectively the data of universal prophylaxis strategy with oral ganciclovir from 2001 to 2004 in our center.We find that though the HCMV infection rate and death rate of prophylaxis group decline to some extent,the extent of descend is not ideal.HCMV pneumonia is still the primary cause of death of renal recipient in early stage in our center and the incidence of HCMV pneumonia didn't declined in prophylaxis group.So,basing on the retrospectively analyzing of the data in our center,we hope to establish a low cost,very simple and easy to be operated program to cut down the rate of HCMV infection,HCMV pneumonia and death, cut down the HCMV correlate complications,and lighten the burden of medical treatment in the early stage after renal transplantation.Part 1.The clinical application and practical value of real-time quantitative PCR in Monitoring HCMV-DNA load of blood and urine in Renal Transplant Recipients.Objective To investigate the practical value of RT-PCR in diagnosing of HCMV infection and the feasibility as the method of dynamic monitoring HCMV-DNA load in pre-empty prophylaxis strategy.Methods 45 recipients who undergone the first time renal transplantation in our center from August 2000 to December 2001 were selected.The HCMV-DNA copies in blood preparation and urine aliquot were tested using RT-PCR every week from 2 weeks to 16 weeks after operation.Simultaneously,the pp65 in blood was tested with two-step immunohistochemistry.The same indexes were tested every 2 weeks from 17 weeks to 24 weeks and every month from 6 months to 12 months after operation.Establish the experiment condition to quantificate HCMV-DNA by RT-PCR(SLAN,FQ PCR).For this process, TaqMan probes,which detect a 89-bp fragment from the HCMV promoter were used.The positive standard was HCMV-DNA load which exceeded 10~3 copies per milliliter.With one positive test result of the blood preparation and urine aliquot,the patient was diagnosed with HCMV infection.During 12 months follow-up,the changes of HCMV-DNA load were monitored continuously,and the characteristic and practical value of RT-PCR on monitoring CMV-DNA load and two-step immunohistological method on testing pp65 antigenaemia in diagnosing active HCMV infections and guiding the antiviral therapy were analyzed.Results Among the 1125 samples,the positive rate was 7.20%(81/1125) through RT-PCR,with a virus load varying between 1.254×10~3 copies /ml and 6.972×10~6 copies/ml.Through pp65 antigenemia analysis, the positive rate was 5.16%(58/1125),with an antigen index between 5/2×10~5 WBC and 12/2×10~5 WBC.The positive rate of patients was 37.80%(17/45) through RT-PCR and 28.89%(13/45) through antigenemia analysis.Cohen's-Kappa test approved that these two methods had a good consistency.The first time of turning into positive through RT-PCR was from 15 to 141 days and the median time of 52 days,while from 31 to 158 days with the median time of 73 days through pp65 antigenemia analysis.The difference of that two methods were statistically significant(p<0.05).6 patients(13.3%) developed into HCMV pneumonia,and the onset time was from 80 to 154 days after transplantation.The HCMV-DNA load of all of these 6 patients began to rise and exceed between 17 and 51 days before onset, but only 4 cases turned into positive before onset.Conclusions Quantitative analysis of HCMV-DNA load displayed good consistency with pp65 antigenaemia analysis,yet it had higher sensitivity.The first time of turning into positive through RT-PCR is earlier than that through pp65 antigenaemia analysis.So RT-PCR is fit for dynamic monitoring HCMV-DNA load and can guide the pre-emptive therapy after kidney transplantation.We must test the HCMV-DNA copies in blood preparation and urine aliquot at the same time and the period is 6 months after operation.Part 2 A retrospectively study about status and therapeutic effect of universal prophylaxis strategy of HCMV infection in early stage after renal transplantation in our hospitalObjective To retrospectively analyze the status of HCMV infection and precaution effect of universal prophylaxis with oral ganciclovir in kidney transplantation recipients during the period of 2001-2004 in our hospital.Methods We analyzed the data of 220 recipients who undergone transplantation in our center from January 2000 to December 2004. All the patients had the follow-up information above 6 months.According to with or without prophylaxis,They were divided into group A(without prophylaxis,89 cases) and group B(universal prophylaxis with oral ganciclovir,131 cases).The universal prophylaxis program was to take ganciclovir(0.5g tid 3 months successively) after renal transplantation.We analyzed the percentage of the recipients that died of HCMV pneumonia in the total died,and compared the incidence of HCMV infection,HCMV pneumonia, acute rejection,other infections,complication,and badness affairs between the two groups.Results The whole incidence of HCMV infection in the two groups was 33.64%(74/220).In group A,the incidence was 41.57%(37/89),the time of taking place was between 17 and 142 days after operation, the median time was 43 days,12 cases developed into HCMV pneumonia and 5 recipients died.In group B,the incidence was 28.24%(37/131), the time of taking place was between 62 and 136 days after operation, the median time was 78 days,15 cases developed into HCMV pneumonia and 2 recipients died.In group A,the incidence of acute rejection was 13.48%(12/89),the whole incidence of infection was 29.31% (26/89),the incidence of the decrease of WBC was 5.62%(5/89),the incidence of hepatic dysfunction was 79.78%(71/89),the incidence of diarrhea was 20.22%(18/89).In group B,the incidence of acute rejection was 11.45%(15/131),the whole incidence of infection was 26.71%(35/131),the incidence of the decrease of WBC was 14.50% (19/131),the incidence of hepatic dysfunction was 74.81%(98/131), the incidence of diarrhea was 16.79%(22/131).Between the 2 groups, the incidence of HCMV infection,death and decrease of WBC was significant difference.The incidence of HCMV pneumonia,acute rejection,hepatic dysfunction and diarrhea wasn't significant difference.From 2001 to 2004,the death rate of HCMV pneumonia in the early stage after renal transplantation showed an ascendant trend.HCMV pneumonia was still the main cause of death in the early stage after renal transplantation.Conclusions HCMV pneumonia is still the main cause of death in the early stage after renal transplantation.The universal prophylaxis strategy with oral ganciclovir can cut down the HCMV infect rate and death rate to some extent,but the decreased degree is not ideal and it can't cut down the incidence of HCMV pneumonia effectively.The therapeutic effect of universal prophylaxis strategy with oral ganciclovir was limited.Part 3 Clinical study of pre-emptive prophylaxis strategy to prevent HCMV pneumonia after renal transplantationObjective To investigate clinical effectiveness and safety of pre-emptive prophylaxis of HCMV pneumonia after renal transplantation by dynamic monitoring HCMV-DNA load with fluorescent quantitative PCR.Methods 242 recipients who performed firstly renal transplantation in our hospital between January 2005 and April 2008 were divided into two groups(test group 127 cases,control group 115 cases) randomly. HMCV-DNA in blood preparation and urine aliquot of recipients in test group were routinely monitored by RT-PCR every week from 2 to 16 weeks and every 2 weeks from 17 to 24 weeks after transplantation. Pre-emptive therapy was initiated with intravenous ganciclovir for 4 weeks when HCMC-DNA load exceeded 10~3 copies/ml in blood preparation or urine aliquot.The dosage was calculated strictly according to creatinine clearance rate.The recipients with clinical symptom who were diagnosed with HCMV pneumonia were treated according to the therapeutic program of HCMV pneumonia.We didn't test the HCMV-DNA in the control group except the clinical symptom of HCMV pneumonia appeared.Those who were diagnosed with HCMV pneumonia were treated with intravenous ganciclovir.We compared the incidence of HCMV pneumonia,complications and adverse event between the 2 groups.Results The incidence of HCMV pneumonia of test group was 6.30% (8/127) and that of control group was 14.78%(17/115).The time of taking place of test group was between 46 and 167 days after operation with the median time was 84 days and that of control group was between 34 and 138 days with the median time was 51 days.The death rate of HCMV pneumonia of test group was 0.79%(1/127) and that of control group was 5.22%(6/115).The ventilator usage of HCMV pneumonia patients of test group was 12.5%(1/8) and that of control group was 58.82%(10/17).The incidence of other pathogen complicated by HCMV pneumonia of test group was 12.5%(1/8) and that of control group was 64.71%(11/17).The incidence ofacute rejection of test group was 5.51%(7/127) and that of control group was 15.65%(18/115).The 1 year survival rate of patients and kidney of test group was 98.42% (125/127) and 97.64%(124/127),and those of control group was 93.04% (107/115) and 91.30%(105/115).The average hospitalization expenses of HCMV pneumonia of test group was￥12864±6526 and that of control group was￥24975±18924.The incidence of hepatic dysfunction of test group was 15.74%(20/127) and that of control group was 29.57% (34/115).The incidence of diarrhea of test group was 10.23%(13/127) and that of control group was 21.73%(25/115).The differences were all statistically significant(P<0.05).The average admission day of HCMV pneumonia patients of test group was 32±14 days and that of control group was 37±29 days.The whole incidence of infection of test group was 27.56%(35/127) and that of control group was 33.91%(39/115).The differences were not statistically significant(P>0.05).The differences of renal function and decrease of WBC between the two group were not statistically significant(P>0.05).There were no severe adverse effects after using intravenous ganciclovir for 4 weeks.Conclusion Pre-empty prophylaxis strategy can prevent HCMV pneumonia in renal transplant recipients,and cut down the incidence, mortality.And pre-empty prophylaxis strategy can improve the 1 year survival rate of patients and kidney.The pre-empty prophylaxis strategy of using intravenous ganciclovir for 4 weeks showed good safety,with he dosage of which was calculated according to the patients' creatinine clearance.