The Laboratory Examination And The Relations With The Lymphatic Cellular Sub-Group Of HCMV Active Infection

HCMV infection can be a major and a critical complication following the organ transplantation.The high rate of death for HCMV infection is the primary factor causing the death of the recipients and the failure of the organ transplantation.HCMV has been considered as a conditional pathogen. The evidence has demonstrated that 50%~90% of people have been infected by HCMV and have been the carriers of its specific antibody.As other herpes virus, HCMV has not demonstrated the obvious pathopoiesis to the individual with the normal immunity, however, it can be activated causing the infection among those carriers with immunity suppression resulted from multi-factors.The recipients are prone to the HCMV infection due to the immunity suppression caused by the using the immunity suppressants in the treatment against the repellency to the implants following the organ transplantations. Thus, the rapid and accurate diagnosis on the HCMV active infection has been the key issue to monitoring the post transplantation infection, providing in time antivirus treatment and judging the future healing process.The routine laboratory examination includes the followings: virus culture and pathology diagnosis, serum HCMV-IgG and HCMV-IgM tests, antigen and HCMV gene identification. According to the demands of the current clinical situation, the main method to diagnose the HCMV active infection relies on the identification of serum HCMV-IgG and HCMV-IgM and IFA to identify CMV-Ag pp65, but there are no many studies and researches focusing on the investigation of the HCMV antigen pp67 and its clinical significance still remains unclear. Meanwhile, the relationship between HCMV infection and cellular immunity state hasn't been entirely known, and the importance of the diagnosis and identification of the HCMV infection on the test of lymphatic cellular sub-group in peripheral blood is still under the argument. Based on the current condition of our laboratory, FCM has been introduced to test CMV-Ag pp65 and NASBA, RT-PCR to identify the HCMV pp67-mRNA. Meanwhile, combined with the situation of the significance of CMV-Ag pp65 and pp67-mRNA to the diagnosis of the HCMV infection, its process and the recovery after transplantation have been evaluated. Moreover, with the test of lymphatic cellular sub-group in peripheral blood and the discussion of the interaction between the patient's immunity state and the HCMV infection, it provides the evidence for the more accurate and effective analysis and understanding for laboratory results in the clinical settings.1.The diagnosis value of CMV-Ag pp65 and pp67-mRNA to the diagnosing the HCMV active infectionTotal 120 samples were collected from transplantation recipients peripheral blood treated with EDTA from 28 recipients between Sep. 2005 and May. 2008 and came from the same recipient at least three times. After one week and one month of the transplantation operation, the peripheral blood about 5~7 ml treated with EDTA was collected from the recipients test immediately. Two groups of ROC to diagnose HCMV were drawn after testing the CMV-Ag pp65 via IFA and pp67-mRNA via NASBA. Comparing with the proportion of two groups of curved lines, it had been found that there were 42 samples out of 120 samples peripheral blood samples in CMV-Ag pp65 positive and 23 samples in pp67-mRNA positive. Among 28 recipients, 9 patients had been found positive in CMV-Ag pp65 and pp67-mRNA (including 3 recipients who were found positive both in CMV-Ag pp65 and in pp67-mRNA at the same time); 8 recipients who were found positive in CMV-Ag pp65 but negative in pp67-mRNA; on the contrary, 2 recipients who were found positive in pp67-mRNA but negative in CMV-Ag pp65; 4 recipients were clinically diagnosed with HCMV desease. There were no any symptoms in the early stage of the HCMV infection and in the latent period. In the early stage of the HCMV infection, the proportion of CMV-Ag pp65 ROC under AUC was 0.9542 and pp67–mRNA ROC under AUC was 0.6611, therefore the sensitivity and specificity of the CMV-Ag pp65 was higher than that of pp67-mRNA. During the periods of symptoms appeared and the late stage of clinical treatment, the proportion of CMV-Ag pp65 ROC under AUC was 0.8300 and the AUC for pp67-mRNA was 0.9232, therefore the sensitivity and specificity of pp67-mRNA was the higher than that of CMV-Ag pp65. In the early stage of the HCMV infection, the AUC for CMV-Ag pp65 was 0.9542 and the AUC for pp67-mRNA was 0.6611. The AUC for CMV-Ag pp65 and pp67-mRNA were 0.8300 and 0.9232 at the late stage after the treatment of one month respectively. It was also suggested by ROC that CMV-Ag pp65>10 positive cell of /2×105WBC was the most significant value for the active HCMV to be diagnosed. 2.Testing CMV-Ag pp65 of the organ transplantation recipients in peripheral blood via FCMThe CMV-Ag pp65 from 145 anticoagulation peripheral blood samples collected from 84 organ transplantation recipients between Sep. 2007 and Mar.2008 had been tested via IFA, and results had shown that the sensitivity was 100% and specificity was 76.0%. Meantime, CMV-Ag pp65 from 16 samples from 9 organ transplantation recipients had been tested via FCM, and the findings indicated that the sensitivity was 100% and specificity was 54.5% . There were no obvious difference of IFA detection and FCM detection by Fisher's exact test(P=0.2520>0.05). 3.Testing pp67-mRNA of the organ transplantation recipients in peripheral blood through Reverse Transcription-PCR technology The total 68 samples were collected from EDTA anticoagulation blood from 38 recipients (male= 24, female=14) who had received kidney transplantation from Oct. 2007 to Jan. 2008. The age for the recipients was between 17 to 73 with the average age of 47. After testing the pp67-mRNA by RT-PCR, the report revealed that 3 samples from 3 patients had demonstrated the positive results with one being diagnosed as HCMV desease(including 2 samples from 2 recipients whose CMV-Ag pp65 were positive and 1 recipients who was diagnosed as HCMV desease). 4.The relations of the HCMV antibody serum status and lymphatic cellular sub-group of the organ transplantation recipients with HCMV active infection Total 27 samples were collected from the peripheral blood from 27 recipients between Sep. 2006 and Feb. 2008 including 3 recipients of the liver transplantation, 20 recipients of the kidney transplantation and 4 recipients of the bone marrow transplantation. In the recipients, 20 patients were male and female take up 7 patients and their ages were between 20 to 54 with the average age of 37. All the recipients demonstrated HCMV-IgG positive and HCMV-IgM negative via. In the detection of CMV-Ag pp65 for 27 samples, the positive results had been reported in 7 samples coming from 7 organ recipients who had clinically been diagnosed of the HCMV active infection. In the analysis the percentage of CD3~+lymphocyte, CD4~+lymphocyte, CD8~+lymphocyte through the CELLQUEST software designed by US, BD Company and calculation the value of CD4~+lymphocyte and CD8~+lymphocyte for each recipients, each group of the statistics had shown as the mean±SD( (x|-)±s). The obviosity of the mean between the groups tested by revising t test. The CD3~+ lymphocyte of the HCMV unactive infection and the HCMV active infection group was (75.96±8.18)% and(67.75±17.39)% respectively, and there was no obvious difference between two groups ( P=0.11>0.05). The CD4~+ lymphocyte of the HCMV unactive infection and the HCMV active infection group was (53.37±4.38)% and( 29.25±9.38 ) % respectively, and the difference between two groups was significant( P=0.0001<0.01). The CD8~+ lymphocyte of the HCMV unactive infection and the HCMV active infection group was (20.73±4.45)% and(36.12±15.60)% respectively, and the difference between two groups was significant( P=0.0003<0.01). The CD4~+ /CD8~+ of the HCMV unactive infection and the HCMV active infection group was (2.64±0.53) and(1.07±0.45)respectively,and the difference between two groups was significant( P=0.0001<0.01).Conclusion:1.There were clinical significance both of CMV-Ag pp65 and of pp67-mRNA. Testing for CMV-Ag pp65 was more suitable for the HCMV active infection at the early stage which was important for in time anti-virus treatment in the clinical settings. While the optimal value to diagnose the active HCMV could be set at CMV-Ag pp65>10 positive cell of /2×105WBC in peripheral blood. The test for pp67-mRNA had been found to be a quick method with the accurate results which could, therefore, be used as the reference criteria for the end of anti-virus treatment. It was significant and valuable as the supplement methods for clinical diagnosis if both of them were combined.2.It had been found that there was no significant difference comparing the specialty between CMV-Ag pp65 testing by FCM and CMV-Ag pp65 testing by IFA.3.RT-PCR could be used for the judgment on pp67-mRNA.4.The single testing for CD3+ lymphocyte production was not valuable for the identification of diagnosis. HCMV antigen out of the body mainly stimulated the CD8~+ lymphocyte proliferate, and there was significant difference between the HCMV unactive infection and the HCMV active infection group in statistics. Thus, when HCMV active infection happened, the entire number and proportion for CD8~+ lymphocyte increased in peripheral blood resulting in the value of CD4~+ lymphocyte and CD8~+ lymphocyte to decrease or even reverse.