Kidney transplantation: evaluation and clinical outcome of 237 recipients at low, medium, high, or strong immunological risk of rejection.

BACKGROUND: Donor-specific antibodies (DSAs) play a fundamental role in kidney transplantation. The identification of DSAs is an essential rejection parameter. PATIENTS AND METHODS: We evaluated a protocol in 237 patients receiving kidneys from living (LDs) and deceased donors (DDs). Recipients were classified as being at low (LR), medium (MR), high (HR), or strong (SR) risk of rejection based on Luminex panel reactive antibody (PRA)-single antigen beads (SABs). Grafts that survived for 1 year were evaluated. RESULTS: Of the 237 transplanted patients, 129 (54.43%) received a kidney from an LD and 108 (45.57%) from a DD. Of 95 LR recipients receiving kidneys from LDs, 2 patients lost the graft due to non-immunological causes. Of 34 MR recipients, 13 had rejection episodes, and 2 lost the graft by AMR and one by cellular rejection (CR). Of 108 recipients receiving a kidney from a DD, 59 (54.63%) were LR, 31 (28.70%) MR, 11 (10.19%) HR, and 7 (6.48%) SR. Twenty of all transplanted recipients lost their grafts; 4 were due to clinical causes, 4 by cellular rejection, and 12 by antibody-mediated rejection (AMR) with PRA-SAB mean fluorescent intensity of 530 to 12,591. One-year graft survival for LD transplanted LR and MR patients was 97.6% and 94.1%, respectively (P = .004). In DD recipients, the LR vs MR SD was P = .011, and for LR vs HR + SR it was P = .001. For MR vs HR+SR no SD was found (P = .323). CONCLUSION: Rejections were detected in 51 patients (21.52%). Graft failure occurred in 16 patients (6.75%). A total of 218 (91.98%) recipients maintained good kidney function after 1 year. This protocol based on fluxogram risk assessment of AMR provided fast and precise immunological evaluation of recipients and donors and stratification by immunological risk of AMR.

Monitoring human cytomegalovirus viral load in peripheral blood leukocytes of renal transplant recipients by a simple limiting dilution-PCR assay.

To assess the clinical relevance of a semi-quantitative measurement of human cytomegalovirus (HCMV) DNA in renal transplant recipients within the typical clinical context of a developing country where virtually 100% of both receptors and donors are seropositive for this virus, we have undertaken HCMV DNA quantification using a simple, semi-quantitative, limiting dilution polymerase chain reaction (PCR). We evaluated this assay prospectively in 52 renal transplant patients from whom a total of 495 serial blood samples were collected. The samples scored HCMV positive by qualitative PCR had the levels of HCMV DNA determined by end-point dilution-PCR. All patients were HCMV DNA positive during the monitoring period and a diagnosis of symptomatic infection was made for 4 of 52 patients. In symptomatic patients the geometric mean of the highest level of HCMV DNAemia was 152,000 copies per 10(6) leukocytes, while for the asymptomatic group this value was 12,050. Symptomatic patients showed high, protracted HCMV DNA levels, whereas asymptomatic patients demonstrated intermittent low or moderate levels. Using a cut-off value of 100,000 copies per 10(6) leukocytes, the limiting dilution assay had sensitivity of 100%, specificity of 92%, a positive predictive value of 43% and a negative predictive value of 100% for HCMV disease. In this patient group, there was universal HCMV infection but relatively infrequent symptomatic HCMV disease. The two patient groups were readily distinguished by monitoring with the limiting dilution assay, an extremely simple technology immediately applicable in any clinical laboratory with PCR capability.

Monitoring human cytomegalovirus viral load in peripheral blood leukocytes of renal transplant recipients by a simple limiting dilution-PCR assay

To assess the clinical relevance of a semi-quantitative measurement of human cytomegalovirus (HCMV) DNA in renal transplant recipients within the typical clinical context of a developing country where virtually 100 per cent of both receptors and donors are seropositive for this virus, we have undertaken HCMV DNA quantification using a simple, semi-quantitative, limiting dilution polymerase chain reaction (PCR). We evaluated this assay prospectively in 52 renal transplant patients from whom a total of 495 serial blood samples were collected. The samples scored HCMV positive by qualitative PCR had the levels of HCMV DNA determined by end-point dilution-PCR. All patients were HCMV DNA positive during the monitoring period and a diagnosis of symptomatic infection was made for 4 of 52 patients. In symptomatic patients the geometric mean of the highest level of HCMV DNAemia was 152,000 copies per 106 leukocytes, while for the asymptomatic group this value was 12,050. Symptomatic patients showed high, protracted HCMV DNA levels, whereas asymptomatic patients demonstrated intermittent low or moderate levels. Using a cut-off value of 100,000 copies per 106 leukocytes, the limiting dilution assay had sensitivity of 100 per cent, specificity of 92 per cent, a positive predictive value of 43 per cent and a negative predictive value of 100 per cent for HCMV disease. In this patient group, there was universal HCMV infection but relatively infrequent symptomatic HCMV disease. The two patient groups were readily distinguished by monitoring with the limiting dilution assay, an extremely simple technology immediately applicable in any clinical laboratory with PCR capability.

Highly sensitive single-step PCR protocol for diagnosis and monitoring of human cytomegalovirus infection in renal transplant recipients.

A multiplex, single-step PCR protocol for the detection of human cytomegalovirus (HCMV) DNA is described. The protocol amplifies regions of the viral LA and IE genes and employs elevated temperatures for both reagent mixing and primer annealing together with product detection by silver staining on polyacrylamide gels. This assay detects one to five HCMV genomes in clinical samples containing up to 100 ng of human DNA, a level of sensitivity equivalent to that of more complex assays involving either nested PCR or postamplification hybridization. As well as being of importance in clinical situations where high-sensitivity qualitative diagnosis is required, this assay is also applicable to the monitoring of HCMV infection in renal transplant recipients. Due to its multiplex format the assay provides quantitative information, in that samples from which a single target is amplified contain on average sevenfold fewer viral genomes per 10(6) leukocytes than those from which both targets are amplified. When weekly blood leukocyte DNA preparations from renal transplant patients were assayed, findings of three consecutive tests in which both HCMV targets were amplified were highly indicative of patients who had developed very high loads of HCMV (100% sensitivity, 88% specificity). We thus show that the same simple PCR assay which permits highly sensitive HCMV diagnosis can also be used for the efficient identification of transplant recipients at risk of clinically significant infection.