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BK virus (BKV) viral load quantification has a distinct role in the clinical control of BKV nephropathy and organ rejection among renal transplant recipients. In this study, it was aimed to compare BKV DNA measurement values performed with two different real-time polymerase chain reaction (PCR) methods and to determine BKV genotypes in renal transplant recipients. Totally, 150 clinical samples tested previously in two different laboratories (Lab-1 and Lab-2) from adult and pediatric renal transplantation patients were included in the study. Fifty plasma samples of 50 different patients from Lab-1, 50 plasma and 50 urine samples of 58 different patients from Lab-2 were included in the study. Viral nucleic acid extraction was performed with automatized systems in Lab-1 and Lab-2 (EZ1, Qiagen, Germany and MagNA Pure 96, Roche Diagnostics, Germany; respectively;). Real-time PCR procedure was carried out in Lab-1 with an amplification mixture of primer, probe sequences targeting VP-1 gene region using RotorGene (Qiagen, Germany) and in Lab-2 with an amplification mixture of primer, probe sequences targeting VP-2 gene region using ABI Prism 7500 (Applied Biosystems, USA). BKV genotyping was performed with multiplex PCR using primer, probe sequences for BKV genotypes I-IV. In both of the laboratories, 82 (54.6%) of the samples were found as positive, 37(24.6%) samples were found as negative and a moderate agreement was found between qualitative results of two real-time PCR methods (ƙ= 0.56, p<0.001). Median viral load values were 4.1 x 104 copies/ml (321-6 x 109) in Lab-1 and 3.3 x 105 copies/ml (224-8.3 x 1010) in Lab-2 for positive samples. According to the lineer regression analysis of quantitative results, moderate (R2= 0.52, p<0.001) and high (R2= 0.88, p<0.001) correlation was found for plasma (n= 52) and urine (n= 30) samples, respectively. Bland-Altman analysis yielded a mean difference of -0.58 log10 for all samples. For plasma samples mean difference was -0.29 log10, while it was -1.1 log10 for urine samples. In all samples, Lab-1 measurements were lower than Lab-2 measurements. A mean difference of -1.1 log10 indicated that the measurement values of Lab-2 were more higher than Lab-1 measurments with an average of 1.1 log10. Supporting this result, 71.9% of the samples had a measurement difference more than 0.5 log 10 and 29.2% of the samples had a measurement difference more than 1 log10. Only 28.1% of the samples were measured within clinically acceptable log difference range (less than 0.5 log10). BKV genotyping was performed only for 74 different patient samples with sufficient copy numbers and genotype I (81.7%), IV (15.5%), II (1.4%), I+IV (1.4%) were detected. When the results were compared; 66.6% (n= 12) of the genotype IV samples had more than 1 log10 and 83.3% of them had more than 0.5 log10 viral load measurement difference. Correlation and linear regression analyzes were insufficient for the comparison ofthe results of the two different tests. It will be appropriate for each center to monitor patients with the same test until the international BKV standard developed by the World Health Organization is optimized. The clinical correlation of the tests is limited to the currently used test. The result of incorrect BKV quantification affects the clinical decision. Measurements less than the actual value will lead to the development of BKV nephropathy, and higher measurements will lead to unnecessary allograft biopsy and unnecessary reduction of immunosuppression.

In spite of the improvements in the clinical management of solid organ transplant (SOT) recipients provided by immunosuppresion and universal prophylaxis, human cytomegalovirus (CMV) infections continue to be one of the most leading causes of morbidity and mortality. Cell-mediated immunity specific to CMV (CMV-CMI) plays an important role in the control of CMV replication. Therefore, monitoring of CMV-specific T-cell response can be used to predict individuals at increased risk of CMV disease. The aim of this study was to investigate the levels of CMV-specific interferon (IFN)-γ producing CD4(+) and CD8(+) T cells in kidney transplant recipients before and after the transplantation, by cytokine flow cytometry. A total of 21 kidney transplant recipients (14 male, 7 female; age range: 18-66 years, mean age: 34.5 ± 9.9) who were all CMV seropositive have been evaluated in the study. Blood samples from the patients were obtained before and at the 1(st), 3(rd) and 6(th) months after transplantation. CMV seropositive healthy kidney donors (n= 20) constituted the control group. The main stages of our procedure were as follows; isolation of peripheral blood mononuclear cells from whole blood, freezing and storing of the samples, later on thawing the samples, ex vivo stimulation of lymphocytes with pooled CMV peptides and counting CMV-specific IFN- producing CD4(+) and CD8(+) T cells by flow cytometry following surface and intracellular cytokine staining. Monitoring of the viral load (CMV-DNA) was performed in 10 days intervals in the first 3 months followed by 3 week intervals until 6 months using COBAS AmpliPrep/COBAS TaqMan CMV test system (Roche Diagnostics, USA). The frequencies of pretransplant CMV-specific IFN-γ producing CD8(+) T cells in patient (3.53 ± 4.35/µl) and control (4.52 ± 5.17/µl) groups were not statistically different (p= 0.266). The difference between the number of virus-specific CD4(+) T cells in patients (8.84 ± 9.56/µl) and those in the control group (8.23 ± 11.98/µl) was at the borderline of significance (p= 0.057). The age and gender of the patients and type of antiviral prophylaxis protocols [valgancyclovir (n= 4); valacyclovir (n= 17)] did not have any significant effect on CMV-CMI (p> 0.05). Similarly, induction therapy administered to four patients did not show any effect on CMV-CMI (p> 0.05). CMV-specific immune responses of patients who received different immunosuppression protocols [tacrolimus + mycophenolate mofetil (MMF) + steroid (n= 17); cyclosporine + MMF + steroid (n= 2); mTOR inhibitor + MMF + steroid (n= 2)] were not different (p> 0.05). The number of CMV-specific CD4(+) T cells in all patients were significantly decreased in the 3rd month compared to the 1st month after the transplantation (p=0.003), indicating a relationship with the period of immunosuppressive therapy. In one of the patients who did not have CMV-specific CD4+ T-cell response but had cytotoxic T-cells (CD8(+) T= 0.6%) before transplantation, CD4(+) T-cell response have developed during monitorization (1.4%, 1.5% and 0.5% in 1st, 3rd and 6th months, respectively), and no viral reactivation was detected. Out of the two patients who had no CD4(+) and CD8(+) T cell response in the 3rd month, one of them developed low level viremia (150 copies/ml) in the 6th month. In this patient the level of CMV-CMI in the 6th month (CD4(+)T + CD8(+)T= 0.9%), have reached higher values than the values obtained before the transplantation (CD4(+) T + CD8(+) T= 0.5%). The viremia was cleared spontaneously in this patient and no antiviral therapy was required. In conclusion, our results suggested that pretransplant and posttransplant monitoring of CMV-specific T-cell responses might be helpful as well as viral load in the clinical management of CMV infection in SOT patients.

Primary BK virus (BKV) infections acquired mainly during childhood are usually asymptomatic. Several studies revealed its seroprevalence in adult population as high as 90% worldwide. Following primary infection, virus persists as latent infection in the urogenital tract. In renal transplant recipients, primary infection and reactivations affect 10% of patients and without treatment, more than half of these patients lose their grafts. The only way of preventing graft loss due to BKV nephropathy (BKVN), seems to monitor BKV infection after transplantation and to diagnose patients developing BKVN during the early period and treat them accordingly. In this study, we analyzed BKV presence in plasma and urine samples with real-time PCR method and evaluated the renal biopsies of pediatric renal transplant recipients after transplantation, retrospectively. A total of 142 children (63 female, 79 male; mean age: 11.7 ± 3.9 years) who had renal transplantation in Akdeniz University Medical Faculty, Antalya, Turkey, between February 2006 and April 2011 were enrolled in the study. After transplantation, peripheral blood and urine samples were collected bi-weekly for the first three months, monthly till the sixth month and every three months thereafter. BKV DNA was additionally screened in patients with unexplained rise in serum creatinine or in patients receiving anti-rejection therapy. In any plasma positivity or during the BKVN therapy, BKV DNA analysis was done bi-weekly. After DNA extraction by automated system, an 83 base pair fragment in VP1 region was amplified. Signal detection for the target region was performed with a TaqMan probe dual-labelled at the 5' end with 6-carboxyfluorescein (FAM) and the 3' end with 6-carboxytetramethylrhodamine (TAMRA). Histopathological examinations of renal biopsies were done with routine histological stains and immunohistochemical staining with monoclonal antibodies directed to SV40 antigen. From 2171 plasma and 1995 urine samples without PCR inhibitors, 442 (20%) (range: 300-4.5 x 10(7) copies/ml; mean: 2.0 x 10(5) ± 2.2 x 10(6) copies/ml) and 800 (40.1%) (range: 300-3 x 10(12) copies/ml; mean: 5.9 x 10(9) ± 1.1 x 10(11) copies/ml) were found positive for BKV DNA, respectively. For 114 (80.3%) patients, at least one urine sample was positive and more than half of those patients (68/114, 59.6%) had viremia. Of the patients, 19.7% (28/142) had viral DNA above 10(4) copies/ml, which was choosen as a cut-off value for its high positive predictive value for BKVN. For all these 28 patients, prior to renal biopsy, immunosupressive treatment was decreased. Cidofovir and/or leflunomid were initiated to nine patients who did not respond to lowered immunosupressive therapy and eight of them had renal biopsy for the confirmation of BKVN. All renal biopsy results were compatible with BKVN. From these nine patients who were receiving cidofovir and/or leflunomid, two lost their grafts because of BKVN. Since viruria is frequently encountered and the viral load is usually in low quantities and transient, it is more appropriate to use blood samples for screening programmes after renal transplantation. The efficacy of antiviral treatment in BKVN could not be evaluated since it was only applied in patients non-responding to lowered immunosuppressive therapy and had decreased renal functions. Multicenter prospective studies are required to enlighten this important issue. Early diagnosis with close monitoring of renal function and viremia, seems to be the most effective way for controlling BKVN.