Prospective study of urine cytology screening for BK polyoma virus replication in renal transplant recipients

Objective:  BK virus (BKV) may be associated with interstitial nephritis in renal transplant recipients and this can lead to irreversible chronic allograft dysfunction. Early diagnosis of BKV nephropathy determines its progress because no specific antiviral therapy exists. Urine cytology, detection of viral DNA in urine or blood and renal biopsy are the main diagnostic tools. The purpose of this study was to evaluate the use of urine cytology for diagnosis of BKV replication in renal graft recipients.
Patients and methods:  We studied 32 de novo renal transplant recipients prospectively with sequential urine samples for a period of 1 year. Thin-Prep methodology was used to prepare the slides. Cytology results were correlated with polymerase chain reaction (PCR) in urine and blood.
Results:  Decoy cells indicative of BKV infection were detected in 14 (7.3%) of the 190 urine samples derived from 11 recipients. In three cases with positive decoy cells, BK viraemia and viruria were simultaneously identified. In a further three cases, BKV active replication was confirmed in urine by both cytology and PCR.
Conclusions:  Urine cytology is an easy and rapid method of detecting decoy cells in cases where renal biopsy is not possible. However, the low incidence of detection of decoy cells in the present study, together with poor correlation with PCR results, questions its sensitivity and specificity in diagnosing BKV reactivation.     

Angiotensinogen,angiotensine converting enzyme and plasminogen activator inhibitor-1 gene polymorphism in chronic allograft dysfunction

Despite dramatic improvements in first-year patient and graft survival rates, chronic allograft dysfunction (CAD) remains the leading cause of late renal allograft loss, while current immunologic strategies have little effect on this condition. The renin–angiotensin system (RAS) plays an important role in progression of chronic renal disease. It was shown that plasminogen activator inhibitor-1 (PAI-1) functions in the RAS. This study investigates the possible links between angiotensinogen (AGT M235T), angiotensin-converting enzyme (ACE) and PAI-1 genotypes with CAD. Assessments of polymorphism were performed in 127 renal allograft recipients (77 with CAD and 50 with normal renal function). Fifty healthy subjects were also considered for comparison. Genotypes were determined using polymerase chain reaction (PCR) sequence-specific primers and PCR followed by restriction fragment length polymorphism analysis. Kidney recipients with CAD had significantly higher frequencies of the TT than the recipients without CAD (P < 0.05). The transplant recipients with CAD also had significantly higher frequencies of the DD genotype than those without CAD (P < 0.05). No significant differences were observed between the allelic and genotypic distributions of PAI-1 polymorphisms. Therefore, determination of AGT M235T and ACE genotypes prior to transplantation may be useful to identify patients who are at risk for chronic renal transplant dysfunction.     

Expression of vimentin and high‐molecular‐weight cytokeratin (clone 34ßE12) in differentiating reactive renal tubular cells from low‐grade urothelial carcinoma cells in voided urine

H. Ohsaki, E. Hirakawa, M. Nakamura, Y. Norimatsu, H. Kiyomoto and R. Haba Expression of vimentin and high‐molecular‐weight cytokeratin (clone 34ßE12) in differentiating reactive renal tubular cells from low‐grade urothelial carcinoma cells in voided urine Objective: Reactive renal tubular cells show features of an atypical repair reaction. Differentiation between reactive renal tubular cells and low‐grade urothelial carcinoma (LG‐UC) cells can therefore be a diagnostic challenge based on morphology alone. In this study, we evaluated the diagnostic utility of vimentin and a high‐molecular‐weight cytokeratin antibody (clone 34ßE12) in differentiating reactive renal tubular cells from LG‐UC. Methods: We evaluated voided urine cytology and surgical specimens from 40 patients with renal disease, and 17 patients with LG‐UC. All slides were stained with vimentin and 34ßE12. Results: In the reactive renal tubular cells in voided urine cytology, vimentin showed strong cytoplasmic staining in 39/40 (97.5%) cases, but all were negative for 34ßE12. LG‐UC cells showed positive staining for 34ßE12 in 3/17 (17.6%) cases, whereas none were positivity for vimentin. The reactive renal tubular cells of histological specimens in the renal disease group demonstrated positive for vimentin in all 40 cases and all were negative for 34ßE12. The LG‐UC group showed abnormal staining for 34ßE12 in 4/17 (23.5%) cases, whereas none were positive for vimentin. Conclusions: Vimentin expression in urine cytology can help to distinguish reactive renal tubular cells from LG‐UC. However, 34ßE12 does not appear to be a useful adjunct to distinguish these two groups in voided urine cytology.     

肾移植术后人微小病毒B19 感染导致纯红细胞增生障碍性贫血

目的:探讨肾移植术后人微小病毒B19感染所致纯红细胞再生障碍性贫血的诊断及其治疗。方法:回顾性分析4例肾移植术后纯红细胞再生障碍性贫血患者的临床特点,诊断方法,治疗过程及预后。结果:两周内在本中心接受肾移植手术的6例患者中,有4例在术后60天内均出现发热、血红蛋白进行性下降等相似症状;综合骨髓穿刺、ELISA方法检测血清特异性IgG、IgM等方法诊断为人微小病毒B19感染;经静脉注射免疫球蛋白、调整免疫抑制方案等综合治疗后,4例患者病情均明显缓解。结论:(1)贫血是肾移植术后患者感染人微小病毒B19的典型临床症状;(2)PCR检测和/或ELISA方法,结合骨髓穿刺及其他实验室指标可诊断人微小病毒感染;(3)静脉注射免疫球蛋白是肾移植术后人微小病毒B19感染导致PRCA的首选治疗方法,病情反复时,再次应用仍然有效。同时予以调整免疫抑制剂方案等综合治疗,可获得理想疗效。     

B‐cell‐activating factor code and human cytomegalovirus infection in renal transplant recipients

The objective of the present study was to explore the correlation between the BAFF signal and HCMV‐TLR activation in RTx recipients complicated by HCMV. Peripheral blood (anticoagulated by EDTA‐Na₂) and urine of 113 RTx recipients were collected; healthy volunteers were controlled. Urine HCMV‐DNA was detected by real‐time PCR. Recipients were classified into a positive group (>10,000 copies/mL urine) and a negative group (<10,000 copies/mL urine). ELISA results showed that sBAFF, sera anti‐HCMV pp65 immunoglobulin (Ig)G antibody, and total IgG all significantly increased in recipients with positive HCMV‐DNA (>10,000 copies/mL urine) (P < 0.05) compared with negative recipients (<10,000 copies/mL urine). In the positive group, HCMV‐DNA copies and total IgG positively correlated with sBAFF (r = 0.988 and 0.625, respectively) (P < 0.05). Luminex assay results suggested that the incidence of anti‐HLA I and II and MICA antibody obviously increased in positive recipients. The expression level of BAFF and BAFF‐R increased in positive recipients. A total of 88 particular genes—involved in TLR signaling pathways, NF‐κB signaling pathways, and cytokine‐cytokine receptor signaling pathways—were detected in real‐time PCR chip assay. A total of 46 genes were differentially expressed greater than two‐fold, and the expression characteristic of BAFF‐R was concordant with FACS results. Our findings are that activation of HCMV would induce or enhance the activation of BAFF code in RTx recipients, which may independently or cooperatively participate in renal allograft injury and decrease the long‐term outcome of renal allografts.     

BKV-DNA and JCV-DNA Co-quantification assay to evaluate viral load in urine and serum

Infections from human polyomaviruses BK and JC (BKV and JCV) occur independently, but concomitant infections and the simultaneous persistence of both viruses have been observed in renal transplant recipients. Several studies have disclosed a correlation between BKV and interstitial nephritis in renal transplant recipients, and an association between JCV and some cases of nephropathy has recently been hypothesized. This article describes the development of a semiquantitative-nested polymerase chain reaction (PCR) assay to simultaneously detect BKV and JCV viral load in urine and serum. The first-round amplification step uses primers that amplify a 385-bp DNA fragment from the “large T antigen” region of both viruses. Samples testing positive in the first step are then run in the second step. In the second-round amplification, different inner primers are used to separately quantify BKV-DNA and/or JCV-DNA. The assay offers several advantages including: (1) rapid submission of clinical samples to screening; (2) verification of the absence of Taq polymerase inhibitors with the use of an internal control; (3) a sensitivity threshold of 10 copies/reaction; and (4) assay running is less labor intensive, cheap, and easy to perform. The assay may be easily used to monitor viral loads versus baseline levels in urine and serum samples from renal transplant recipients to detect those at risk of BKV- or JCV-related nephropathy, and to monitor their response to immunosuppression reduction therapy if it occurs. This paper is dedicated to the memory of Prof. Giorgio Cavallo.     

Infectious complications associated with renal transplantation: an analysis of risk factors.

To assess the multiple risk factors reported to be associated with onset of serious bacterial, fungal, viral, and protozoal infections in renal allograft recipients, a retrospective study of all renal transplantations performed at Yale-New Haven Medical Center from the inception of the transplantation program in December, 1967, to December, 1975, was undertaken. Ninety-six renal allograft transplants in 85 patients were available for evaluation during this study period. Renal allograft recipients were evaluated for incidence of infection from time of transplantation until transplant nephrectomy, death, or January 1, 1976. All infections were characterized by type of infection, organism, site, and time of onset post-transplantation. Recipients with infections were also evaluated for their donor type, living-related or cadaveric, age at time of transplantation, granulocytopenia, corticosteroid therapy, and rejection episodes. There were 215 infections, 92 of which were defined as serious, in 78 of the 96 renal allograft recipients. Eighteen renal allograft recipients had no infections. Granulocytopenia, but not rejection, correlated with serious infections at some time in the patient''s course. However, no significant temporal relationship between serious infections and episodes of granulocytopenia or rejection could be established. Mortality rate and incidence of serious infection was higher in the group receiving high dose corticosteroid therapy compared with the group receiving lower doses of corticosteroids. The mortality rate in these 85 transplant recipients was 33%. Seventy-four percent of these deaths were directly related to infection (24% of 85 patients).     

Renal Transplant Recipients Treated with Calcineurin-Inhibitors Lack Circulating Immature Transitional CD19+CD24hiCD38hi Regulatory B-Lymphocytes

BackgroundCD19⁺CD24^hiCD38^hi transitional immature B-lymphocytes have been demonstrated to play an important role in regulating the alloimmune response in transplant recipients. Here, we analyzed the effect of calcineurin inhibition on these peripherally circulating regulatory B-cells (Breg) in renal transplant recipients receiving cyclosporine A (CsA) or tacrolimus.MethodsPBMCs from healthy subjects (HS) (n = 16) and renal transplant recipients (n = 46) were isolated. Flow cytometry was performed for CD19, CD24, CD38 and IL-10 either after isolation or after 72 hours of co-culture in presence of PMA/Ionomycin and TLR9-ligand in presence or absence of increasing concentrations of tacrolimus or CsA.ResultsThe amount of CD19⁺ B-cells among lymphocytes was ∼9.1% in HS, ∼3.6% in CsA (n = 11, p<0.05) and ∼6.4% in TAC (n = 35, p<0.05) treated patients. Among B-cells, a distinct subset of Breg was found to be 4.7% in HS, 1.4% in tacrolimus treated patients and almost blunted in patients receiving CsA. Similarily, ∼4% of B-cells in HS and even fewer in CsA or tacrolimus treated patients produced IL-10 (0.5% and 1.5%, p<0.05) and this was confirmed both in non-transplanted CsA-treated healthy subjects and in in vitro co-culture experiments. Among 29 patients with <1% of Breg, 9 cases (31%) displayed an allograft rejection in contrast to only one case of rejection (6%) among 17 patients with >1%.ConclusionCalcineurin inhibitors reduce number and IL-10 production of Bregs in the peripheral circulation of both renal transplant recipients and non-transplanted healthy subjects. CNI induced Breg reduction is not restricted to a solid organ transplant setting and is not mediated by co-medication with steroids or MPA. A low proportion of Breg cells is associated with an elevated frequency of allograft rejection events.     

肾移植受者生活质量和健康素养现状调查及影响因素分析

目的:调查肾移植受者生活质量(QOL)、健康素养(HL)现状,并分析其QOL、HL的影响因素。方法:选择我院2015年1月～2016年1月收治的369例肾移植受者为研究对象,采用自制问卷结合病历信息的方式收集入选患者的临床资料,分别采用健康状况调查简表(SF-36)、中文版成人快速健康素养评估量表(REALAM-T)对肾移植受者的QOL、HL的现状进行调查,并分析肾移植受者QOL、HL的影响因素。结果:369例肾移植受者QOL评分为(561.08±54.95)分,HL评分为(62.75±5.26)分,其中288例(78.05%)患者处于HL充足水平,56例(15.18%)患者处于HL临界水平,25例(6.78%)患者处于HL缺乏水平。单因素分析结果显示,不同婚姻状况、家庭人均月收入、文化程度、费用支付方式、移植肾来源、移植术后时间的肾移植受者QOL评分比较差异有统计学意义(P0.05);不同家庭人均月收入、文化程度、费用支付方式、移植肾来源、移植术后时间的肾移植受者HL评分比较差异有统计学意义(P0.05)。多元线性回归分析显示,家庭人均月收入、费用支付方式、移植肾来源、移植术后时间是肾移植受者QOL的影响因素(P0.05),文化程度、移植肾来源、移植术后时间是肾移植受者HL的影响因素(P0.05)。结论:肾移植受者QOL较差,HL整体不高,家庭人均月收入、费用支付方式、移植肾来源、移植术后时间是肾移植受者QOL的影响因素,文化程度、移植肾来源、移植术后时间是肾移植受者HL的影响因素,临床应根据以上因素采取针对性的措施,以提高肾移植受者的QOL和HL。     

Association of common variants of vascular endothelial growth factor and interleukin-18 genes with allograft survival in renal transplant recipients of North India

Increased vascular endothelial growth factor (VEGF) production promotes enhanced endothelial permeability, enhanced leukocyte migration into the allograft, thereby leading to a clinically recognized rejection episode. Interleukin-18 (IL-18), a potent proinflammatory cytokine, may also be involved in mechanisms of kidney allograft rejection. The present study was, therefore, undertaken to investigate the association of functional polymorphisms in VEGF (2578C>A, 1154A>G) and IL-18 (607C>A, 137G>C) genes with risk of allograft rejection in renal transplant recipients of North India. Two hundred renal transplant recipients, 150 matched recipients-donors, and 200 unrelated healthy individuals were genotyped by amplification refractory mutation specific polymerase chain reaction and by polymerase chain reaction-restriction fragment length polymorphism. Variant allele VEGF 1154A>G (p?=?0.56; odds ratio [OR]?=?1.32) and variant allele (p?=?0.004, OR?=?1.54) and variant genotype (p?=?0.007, OR?=?3.26) of IL-18 607C>A, GC of IL-18 137G>C (p?=?0.043, OR?=?0.63) were significantly different in healthy individuals as compared with the patients with renal transplant. When 114 nonrejectors were compared with 36 rejectors (150 recipients) for association with allograft rejection, significant association was observed in heterozygous genotype of VEGF 2578C>A (p?=?0.033), VEGF 1154A>G (p?=?0.024). In IL-18 137G>C, CC genotype, C allele showed protective association with allograft rejection. Kaplan-Meier analysis indicated a higher mean time for first rejection episode in CA genotype carriers (31 months) as compared with AA (29 months) for VEGF 2578C>A (log p?=?0.035). In VEGF, the haplotypes A-A and A-G (2578-1154) were associated with reduced risk and in IL-18 607A-137G, they were associated with high risk for allograft rejection. This observation suggests these polymorphisms are an ideal marker for prediction of pretransplant allograft outcome.     

Immunological biomarkers of tolerance in human kidney transplantation: An updated literature review

The half-life of transplanted kidneys is <10 years. Acute or chronic rejections have a negative impact on transplant outcome. Therefore, achieving to allograft tolerance for improving long-term transplant outcome is a desirable goal of transplantation field. In contrast, there are evidence that distinct immunological characteristics lead to tolerance in some transplant recipients. In contrast, the main reason for allograft loss is immunological responses. Various immune cells including T cells, B cells, dendritic cells, macrophages, natural killer, and myeloid-derived suppressor cells damage graft tissue and, thereby, graft loss happens. Therefore, being armed with the comprehensive knowledge about either preimmunological or postimmunological characteristics of renal transplant patients may help us to achieve an operational tolerance. In the present study, we are going to review and discuss immunological characteristics of renal transplant recipients with rejection and compare them with tolerant subjects.     

Prospective study of BKV nephropathy in 117 renal transplant recipients

Nephropathy caused by poliomavirus (BKVAN) in transplant recipients is responsible for the loss of the transplanted organ. In this study we suggest a non-invasive diagnostic protocol for the early identification of BKVAN during follow-up treatments. In 117 kidney transplant recipients follow-up was performed every three months during a two year period after transplantation and a positive screening result was confirmed and assessed by quantitative assays (BKV DNA load in plasma and urine). The definitive diagnosis of BKV requires allograft biopsy. Of the 117 patients 4 had BKVAN (3.4%), and the consequential reduction of immunosuppression improved kidney function and plasma clearance of the virus was achieved.     

Changes in Surgical Site Infections after Living Donor Liver Transplantation

Surgical site infections (SSIs) are a major threat for liver transplant recipients. We prospectively studied SSIs after living donor liver transplantation (LDLT) at Kyoto University Hospital from April 2001 to March 2002 (1^st period) and from January 2011 to June 2012 (2^nd period). We investigated the epidemiology of SSIs after LDLT and determined the differences between the two periods. A total of 129 adult recipients (66 during the 1^st period and 63 during the 2^nd period) and 72 pediatric recipients (39 and 33) were included in this study. The SSI rates for each period were 30.3% (1^st period) and 41.3% (2^nd period) among the adult recipients and 25.6% and 30.3% among the pediatric recipients. The overall rates of 30-day mortality among adult transplant recipients with SSIs were 10.0% (1^st period) and 3.9% (2^nd period). No pediatric recipient died from SSIs after LDLT in either period. The incidence of Enterococcus faecium increased from 5.0% to 26.9% in the adults and from 10.0% to 40.0% in the pediatric patients. Extended-spectrum β-lactamase-producing Enterobacteriaceae were emerging important isolates during the 2^nd period. For this period, a univariate analysis showed that ABO incompatibility (P = 0.02), total operation duration (P = 0.01), graft-to-recipient body weight ratio (GRWR [P = 0.04]), and Roux-en-Y biliary reconstruction (P<0.01) in the adults and age (P = 0.01) and NHSN risk index (P = 0.02) in the children were associated with SSI development. In a multivariate analysis, lower GRWR (P = 0.02) and Roux-en-Y biliary reconstruction (P<0.01) in the adults and older age (P = 0.01) in the children were independent risk factors for SSIs during the 2^nd period. In conclusion, SSIs caused by antibiotic resistant bacteria may become a major concern. Lower GRWR and Roux-en-Y biliary reconstruction among adult LDLT recipients and older age among pediatric LDLT recipients increased the risk of developing SSIs after LDLT.     

HLA-DRw6 as a risk factor for active cytomegalovirus but not for herpes simplex virus infection after renal allograft transplantation.

To study genetically determined susceptibility to cytomegalovirus and herpes simplex virus infections in patients given renal transplants a prospective study was performed of 68 consecutive patients receiving their first cadaveric kidney allograft. The recipients positive for HLA-DRw6 showed a significantly increased incidence of active cytomegalovirus infection as early as the 10th week after transplantation (p less than 0.05). No relation with other human leucocyte antigens was found, nor did a correlation exist between HLA typing and the incidence of herpes simplex virus infections. Furthermore, recipients positive for HLA-DRw6 with secondary cytomegalovirus infections excreted infectious virus more often (p less than 0.01) and showed more clinical symptoms (p less than 0.01) than a comparable group of recipients negative for HLA-DRw6. These observations may have practical implications for the treatment of patients who have had renal transplant operations.     

Assessment of the status of cell-mediated immunity in cytomegalovirus-infected renal allograft recipients.

Renal allograft recipients are unusually susceptible to cytomegalovirus (CMV) infections. Since humoral immunity to CMV is uncompromised in these patients, it was felt desirable to assess the competence of cell mediated immunity (CMI). Several parameters were used. On skin testing with candida, SK-SD, mumps, and PPD-5 antigens, 80.0% of patients and 5.0% of controls were unreactive. T-lymphocyte ratios (SRBC rosette test) were 18.7% in transplant patients, vs. 40.3% in controls. These differences are statistically significant. Lymphocyte stimulation assay ([³H] thymidine uptake) was developed to study CMI to CMV. Lymphocytes from all the normal seropositive subjects (10) had increased [³H]thymidine uptake on exposure to CMV antigens. There was no antigen specific stimulation of lymphocytes from the seronegative controls (five). Six of nine (67.7%) CMV infected renal allograft recipients, studied six or more months post-transplantation, had no evidence of CMI to CMV by this assay.     

The prevalence of immunologic injury in renal allograft recipients with de novo proteinuria

Post-transplant proteinuria is a common complication after renal transplantation; it is associated with reduced graft and recipient survival. However, the prevalence of histological causes has been reported with considerable variation. A clinico-pathological re-evaluation of post-transplant proteinuria is necessary, especially after dismissal of the term "chronic allograft nephropathy," which had been considered to be an important cause of proteinuria. Moreover, urinary protein can promote interstitial inflammation in native kidney, whether this occurs in renal allograft remains unknown. Factors that affect the graft outcome in patients with proteinuria also remain unclear. Here we collected 98 cases of renal allograft recipients who developed proteinuria after transplant, histological features were characterized using Banff scoring system. Cox proportional hazard regression models were used for graft survival predictors. We found that transplant glomerulopathy was the leading (40.8%) cause of post-transplant proteinuria. Immunological causes, including transplant glomerulopathy, acute rejection, and chronic rejection accounted for the majority of all pathological causes of proteinuria. Nevertheless, almost all patients that developed proteinuria had immunological lesions in the graft, especially for interstitial inflammation. Intraglomerular C3 deposition was unexpectedly correlated with the severity of proteinuria. Moreover, the severity of interstitial inflammation was an independent risk factor for graft loss, while high level of hemoglobin was a protective factor for graft survival. This study revealed a predominance of immunological parameters in renal allografts with post-transplant proteinuria. These parameters not only correlate with the severity of proteinuria, but also with the outcome of the graft.     

High MCM3 expression is an independent biomarker of poor prognosis and correlates with reduced RBM3 expression in a prospective cohort of malignant melanoma

Background

After the introduction of novel effective immunosuppressive therapies, kidney transplantation became the treatment of choice for end stage renal disease. While these new therapies lead to better graft survival, they can also cause a variety of complications. Only small series or case reports describe pulmonary pathology in renal allograft recipients on mTOR inhibitor inclusive therapies. The goal of this study was to provide a systematic review of thoracic biopsies in kidney transplant recipients for possible association between a type of immunosuppressive regimen and pulmonary complications.

Methods

A laboratory database search revealed 28 of 2140 renal allograft recipients (18 males and 10 females, 25 to 77 years old, mean age 53 years) who required a biopsy for respiratory symptoms. The histological features were correlated with clinical findings including immunosuppressive medications.

Results

The incidence of neoplasia on lung biopsy was 0.4% (9 cases), which included 3 squamous cell carcinomas, 2 adenocarcinomas, 1 diffuse large B-cell lymphoma, 1 lymphomatoid granulomatosis, and 2 post transplant B-cell lymphoproliferative disorders. Diffuse parenchymal lung disease was identified in 0.4% (9 cases), and included 5 cases of pulmonary hemorrhage, 3 cases of organizing pneumonia and 1 case of pulmonary alveolar proteinosis. Five (0.2%) cases showed histological features indicative of a localized infectious process. Patients on sirolimus had neoplasia less frequently than patients on other immunosuppressive combinations (12.5% vs. 58.3%, p = 0.03). Lung biopsies in 4 of 5 patients with clinically suspected sirolimus toxicity revealed pulmonary hemorrhage as the sole histological finding or in combination with other patterns.

Conclusions

Our study documents a spectrum of neoplastic and non-neoplastic lesions in renal allograft recipients on current immunosuppressive therapies. Sirolimus inclusive regimens are associated with increased risk of pulmonary toxicity but may be beneficial in cases of posttransplant neoplasia.

Virtual Slides

The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/3320012126569395.     

Development and validation of multiplex real-time PCR assays for rapid detection of cytomegalovirus,Epstein-Barr virus,and polyomavirus BK in whole blood from transplant candidates

Transplant recipients are more susceptible to bacterial and viral infections. Cytomegalovirus (CMV), Epstein-Barr virus (EBV), and polyomavirus BK (BK) are risk factors for graft dysfunction. All three of them are latent viruses that can cause serious disease in immunocompromised patients. Mainly qualitative PCR tests are required for diagnosis and quantitative monitoring, which are used to follow the response to transplantation. We developed a multiplex real-time PCR (qPCR) method to detect these viruses during blood screenings of transplant recipients. We also validated analytical and clinical performance tests using the developed multiplex qPCR. The limit of detection (LOD) was 100, 125, and 183 copies/ml for CMV, EBV, and BK, respectively. These results had high linearity (R² = 0.997) and reproducibility (CV range, 0.95–2.38%, 0.52–3.32%, and 0.31–2.45%, respectively). Among 183 samples, we detected 8 samples that were positive for CMV, while only 6 were positive for EBV, and 3 were positive for BK. Therefore, the viral infection prevalence in transplant candidates was 4.40% for CMV, 3.29% for EBV, and 1.64% for BK. This multiplex qPCR method should be used widely for diagnosing and monitoring latent viral infections in transplant recipients.     

The ubiquity and impressive genomic diversity of human skin papillomaviruses suggest a commensalic nature of these viruses

Human papillomaviruses (HPV) are epitheliotropic viruses, with some types suggested to be associated with skin cancer. In this study, swab samples collected from five different sites on the skin of renal transplant recipients, dialysis patients, and age- and sex-matched healthy controls were analyzed for HPV DNA by a newly designed PCR test. Most individuals were found to have asymptomatic HPV infections; more specifically, 94% of the renal transplant patients, 82% of the dialysis patients, and 80% of the healthy controls were positive for HPV DNA. The multiplicity of the HPVs detected was astounding: 20 previously described and 30 putatively new types were identified by cloning and sequencing of 33 samples from 13 individuals. These results demonstrate that normal human skin harbors an array of papillomaviruses, most of them previously unknown.     

Arterial elasticity imaging: comparison of finite-element analysis models with high-resolution ultrasound speckle tracking

Background

To resolve the current shortage of donor hearts, we established the Adonhers protocol. An upward shift of the donor age cut-off limit (from the present 55 to 65 years) is acceptable if a stress echo screening on the candidate donor heart is normal. This study aimed to verify feasibility of a "second opinion" of digitally transferred images of stress echo results to minimize technical variability in selection of aged donor hearts for heart transplant.

Methods

The informatics infrastructure was created for a core lab reading with a second opinion from the Pisa stress echo lab. To test the system, simulation standard stress echo cineloops were sent digitally from 5 peripheral labs to the central core lab. Starting January 2009, real marginal donor stress echos were sent via internet to the central core echo lab, Pisa, for a second opinion before heart transplant.

Results

In the simulation protocol, 30 dipyridamole stress echocardiograms were sent from the five peripheral echo labs to the central core lab in Pisa. Both the echo images and reports were correctly uploaded in the web system and sent to the core echo lab; the second opinion evaluation was obtained in all cases (100% feasibility). In the transplant protocol, eight donor cases were sent to the Pisa core lab for the second opinion protocol, and six of them were transplanted in marginal recipients.

Conclusions

Second-Opinion Stress Tele-Echocardiography can effectively be performed in a network aimed to safely expand the heart donor pool for heart transplant.