Characterization of Human Cytomegalovirus Genome Diversity in Immunocompromised Hosts by Whole-Genome Sequencing Directly From Clinical Specimens.

Background: Advances in next-generation sequencing (NGS) technologies allow comprehensive studies of genetic diversity over the entire genome of human cytomegalovirus (HCMV), a significant pathogen for immunocompromised individuals. Methods: Next-generation sequencing was performed on target enriched sequence libraries prepared directly from a variety of clinical specimens (blood, urine, breast milk, respiratory samples, biopsies, and vitreous humor) obtained longitudinally or from different anatomical compartments from 20 HCMV-infected patients (renal transplant recipients, stem cell transplant recipients, and congenitally infected children). Results: De novo-assembled HCMV genome sequences were obtained for 57 of 68 sequenced samples. Analysis of longitudinal or compartmental HCMV diversity revealed various patterns: no major differences were detected among longitudinal, intraindividual blood samples from 9 of 15 patients and in most of the patients with compartmental samples, whereas a switch of the major HCMV population was observed in 6 individuals with sequential blood samples and upon compartmental analysis of 1 patient with HCMV retinitis. Variant analysis revealed additional aspects of minor virus population dynamics and antiviral-resistance mutations. Conclusions: In immunosuppressed patients, HCMV can remain relatively stable or undergo drastic genomic changes that are suggestive of the emergence of minor resident strains or de novo infection.

A rolling circle amplification screen for polyomaviruses other than BKPyV in renal transplant recipients confirms high prevalence of urinary JCPyV shedding.

OBJECTIVES: Multiple novel human polyomaviruses (HPyVs) have been discovered in the last few years. These or other, unknown, nephrotropic HPyVs may potentially be shed in urine. METHODS: To search for known and unknown HPyVs we investigated BKPyV-negative urine samples from 105 renal transplant recipients (RTR) by rolling circle amplification (RCA) analysis and quantitative JCPyV PCR. Clinical data was analysed to identify risk factors for urinary polyomavirus shedding. RESULTS: In 10% (11/105) of the urine samples RCA with subsequent sequencing revealed JCPyV, but no other HPyV sequences. Using quantitative JCPyV PCR, 24% (25/105) of the samples tested positive. Overall sensitivities of RCA of 44% (11/25) in detecting JCPyV in JCPyV DNA-positive urine and 67% (10/15) for samples with JCPyV loads >10,000 copies/ml can be assumed. Despite frequent detectable urinary shedding of JCPyV in our cohort, this could not be correlated with clinical risk factors. CONCLUSION: Routine urinary JCPyV monitoring in BKPyV-negative RTR without suspected polyomavirus-associated nephropathy might be of limited diagnostic value. As RCA works in a sequence-independent manner, detection of novel and known polyomaviruses shed in sufficient quantities is feasible. High-level shedding of HPyVs other than BKPyV or JCPyV in the urine of RTR is unlikely to occur.

Resistant hypertension: baroreflex stimulation as a new tool.

Uncontrolled hypertension remains a significant public health challenge. In recent years, a new baroreflex stimulator has been used to treat these patients. Initial observations suggest that the electrical field stimulation of carotid baroreceptors acutely attenuates sympathetic activation of the vasculature, heart and kidney while augmenting cardiac vagal regulation. During the long-term treatment an average blood pressure (BP) drop of 30-40/15-25 mmHg was observed with a responder rate (>10 mmHg reduction in BP) of up to 80% after 1 year of treatment. Some of this effect can be explained by a 'placebo' effect as suggested by the double-blind Pivotal Trial. The complication rate with the first generation device was 20-30%. With a second generation device, these problems have been reduced to <10%. Even though additional data from controlled clinical trials will be required before more widespread use can be recommended, this treatment option is now approved in Europe for the treatment of severe resistant hypertension and is performed in selected centres with experienced vascular surgeons and hypertension specialists.

Risk Factors Influencing the Outcomes of Kidney Re-Transplantation.

BACKGROUND Our kidney transplant waitlist includes 20% re-transplantations (TX2). Knowing what to expect is a clinical obligation. MATERIAL AND METHODS We compared graft and patient survival of all 162 TX2 patients, transplanted 2000 to 2009, with 162 patients after first transplantation (TX1) matched for age, sex, living/non-living donation, and transplantation date. Patient follow-up was 10 years. RESULTS TX2 graft and patient survivals were inferior to TX1 (p<0.001 and p=0.047). TX2 patients had a longer cumulative dialysis vintage, more human leucocyte antigen (HLA) mismatches, more panel-reactive HLA antibodies, more often received induction therapy with rabbit-antithymocyte globulin (rATG), and had a lower body mass index (all p<0.05). Death from infection and graft failure by rejection was more frequent after TX2 (both p<0.05) but not after TX1. Multivariable Cox regression analysis revealed that both cohorts had graft failure and death risk associated with infection and cardiovascular disease, and graft failure by humoral rejection. However, only TX2 patients had an additional risk of graft failure with early inferior graft function and of patient death with ≥2 comorbidities. Moreover, Kaplan-Meier analysis showed that TX2 and not TX1 patients had a lower graft and patient survival associated with infection and with ≥2 comorbidities (all p<0.05). CONCLUSIONS Re-transplantation is associated with worse graft outcomes mainly because of immunologic and graft-quality reasons, although the high number of comorbidities and infection severities aside from cardiovascular disease drive mortality. The more frequent rATG induction of TX2 patients could promote infection by enhancing immunosuppression. By addressing comorbidities, outcomes could possibly be improved.

Whole-Genome Approach to Assessing Human Cytomegalovirus Dynamics in Transplant Patients Undergoing Antiviral Therapy.

Human cytomegalovirus (HCMV) is the most frequent cause of opportunistic viral infection following transplantation. Viral factors of potential clinical importance include the selection of mutants resistant to antiviral drugs and the occurrence of infections involving multiple HCMV strains. These factors are typically addressed by analyzing relevant HCMV genes by PCR and Sanger sequencing, which involves independent assays of limited sensitivity. To assess the dynamics of viral populations with high sensitivity, we applied high-throughput sequencing coupled with HCMV-adapted target enrichment to samples collected longitudinally from 11 transplant recipients (solid organ, n = 9, and allogeneic hematopoietic stem cell, n = 2). Only the latter presented multiple-strain infections. Four cases presented resistance mutations (n = 6), two (A594V and L595S) at high (100%) and four (V715M, V781I, A809V, and T838A) at low (<25%) frequency. One allogeneic hematopoietic stem cell transplant recipient presented up to four resistance mutations, each at low frequency. The use of high-throughput sequencing to monitor mutations and strain composition in people at risk of HCMV disease is of potential value in helping clinicians implement the most appropriate therapy.

Viral Origin, Clinical Course, and Renal Outcomes in Patients With BK Virus Infection After Living-Donor Renal Transplantation.

BACKGROUND: BK virus (BKV) nephropathy remains the main cause of renal graft loss after living-donor renal transplantation. The aim of the study was to investigate the source and factors influencing the course of BKV infection. METHODS: We investigated 214 living donor-recipient pairs. Urine and blood of donors and recipients were tested by qPCR for the presence of BKV DNA before and after transplantation; genotyping of BKV subtypes was performed. RESULTS: Eighty-five recipients (40%) had posttransplant BK viruria including 61 with additional viremia and 22 with nephropathy. Pretransplant urinary BKV shedding of donor or recipient was a significant risk factor for posttransplant viruria and viremia (OR, 4.52; CI, 2.33-8.77; P < 0.0001) and nephropathy (OR, 3.03; CI, 1.16-7.9; P = 0.02). In the BKV nephropathy group, urine and blood became BKV positive earlier than in the group with viruria and viremia. Renal function was worse in BKV-nephropathy compared with BKV-negative patients beginning at transplantation. Comparing BKV subtypes of donor and recipient before with the subtype of the infected recipient after transplantation, donor-derived transmission was identified in 24 of 28 corresponding pairs. BKV subtype IV had a higher prevalence in recipients with BKV nephropathy than in those with viruria and viremia (P = 0.045). CONCLUSIONS: Pretransplant urinary BKV shedding of donor and recipient is a risk for posttransplant infection. Donor-derived BKV transmission is an important mode of infection. BKV subtype IV may be one of the viral determinants. Early BKV positivity of urine and blood indicates later BKV nephropathy. Decreased renal function may favor BKV infection.

Acute Response to Unilateral Unipolar Electrical Carotid Sinus Stimulation in Patients With Resistant Arterial Hypertension.

Bilateral bipolar electric carotid sinus stimulation acutely reduced muscle sympathetic nerve activity (MSNA) and blood pressure (BP) in patients with resistant arterial hypertension but is no longer available. The second-generation device uses a smaller unilateral unipolar disk electrode to reduce invasiveness while saving battery life. We hypothesized that the second-generation device acutely lowers BP and MSNA in treatment-resistant hypertensive patients. Eighteen treatment-resistant hypertensive patients (9 women/9 men; 53±11 years; 33±5 kg/m(2)) on stable medications have been included in the study. We monitored finger and brachial BP, heart rate, and MSNA. Without stimulation, BP was 165±31/91±18 mm Hg, heart rate was 75±17 bpm, and MSNA was 48±14 bursts per minute. Acute stimulation with intensities producing side effects that were tolerable in the short term elicited interindividually variable changes in systolic BP (-16.9±15.0 mm Hg; range, 0.0 to -40.8 mm Hg; P=0.002), heart rate (-3.6±3.6 bpm; P=0.004), and MSNA (-2.0±5.8 bursts per minute; P=0.375). Stimulation intensities had to be lowered in 12 patients to avoid side effects at the expense of efficacy (systolic BP, -6.3±7.0 mm Hg; range, 2.8 to -14.5 mm Hg; P=0.028 and heart rate, -1.5±2.3 bpm; P=0.078; comparison against responses with side effects). Reductions in diastolic BP and MSNA (total activity) were correlated (r(2)=0.329; P=0.025). In our patient cohort, unilateral unipolar electric baroreflex stimulation acutely lowered BP. However, side effects may limit efficacy. The approach should be tested in a controlled comparative study.

Ultrasound findings in EHEC-associated hemolytic-uremic syndrome and their clinical relevance.

PURPOSE: Hemolytic-uremic syndrome (HUS) and acute kidney injury (AKI) after infection with Shiga toxin-producing E. coli (EHEC) are clinically important complications. We present a retrospective analysis of abdominal ultrasound findings in patients with HUS caused by EHEC O104:H4 (n = 41). METHODS: We assessed intrarenal resistance indices and quantitated kidney parenchymal density by the kidney/liver intensity ratio using computer-based image analysis. Findings in EHEC-HUS were compared to those in AKI due to other reasons (n = 60) and 19 healthy volunteers. RESULTS: Kidneys in EHEC-HUS patients showed severe morphologic changes with striking parenchymal echogenicity. Renal resistance index was increased in HUS (0.80 ± 0.08) compared to patients with AKI due to glomerulopathy (0.69 ± 0.08, p < 0.001) or patients with other causes of AKI (0.74 ± 0.10, p < 0.01). Parenchymal density was increased in EHEC-HUS (1.39 ± 0.35) compared to AKI due to glomerulopathy (1.18 ± 0.20, p < 0.05), other causes of AKI (1.12 ± 0.21, p < 0.001) and healthy controls (0.86 ± 0.16, p < 0.001). Patients with atypical HUS showed increased parenchymal density (1.43 ± 0.37), similar to those with EHEC-HUS. EHEC-HUS patients who required dialysis treatment had higher parenchymal density (1.58 ± 0.08) compared to those without dialysis (1.14 ± 0.05, p = 0.0004). Extrarenal findings in EHEC-HUS included hepatomegaly (45%), splenomegaly (39%), ascites (84%) and pleural effusions (84%). CONCLUSIONS: Patients with EHEC-HUS had a characteristic constellation of morphologic abnormalities on ultrasound examination, indicating that the effects of HUS are not limited to the kidney but include multiple organs, possibly mediated through systemic capillary leakage. Assessment of parenchymal echogenicity contributes to the differential diagnosis of AKI. Parameters reflecting renal perfusion correlated with severity of disease and thus may have prognostic value in future patient evaluation.

Donor origin of BKV replication after kidney transplantation.

BACKGROUND: BK virus associated nephropathy (BKVN) leads to renal allograft dysfunction and loss. However, it is still unclear whether BKV replication in the transplant recipient is a result of reactivation in the recipient's native kidneys or whether BKV originates from the donor kidney. STUDY DESIGN: Urine of 249 donor/recipient pairs was investigated for the presence of BKV-DNA by qPCR before living transplantation (Tx) and consecutively after Tx. In BKV positive samples, the VP1 typing region (TR) and, in case of the presence of sufficient amount of DNA, the complete VP1 gene, the NCCR and a fragment of the Large T-antigen were sequenced and compared between donors and corresponding recipients before and after Tx. RESULTS: In 20 pairs, sequencing of the BKV TR succeeded in donors and corresponding recipients after Tx. The derived sequences were completely identical in donor and post-Tx recipient samples. For comparison, identical TR sequences were detected in only 24% of 1068 randomly assembled pairs. This difference was statistically highly significant (p<0.0001, Fisher's exact test). Furthermore, all VP1, Large T-antigen and NCCR BKV sequences were also identical between donors and corresponding post-Tx recipients. In two of the 20 donor/recipient pairs, VP1 TR sequencing was also successful from the recipient before Tx. In both cases the sequence differed from the sequence detected in donor and recipient after Tx giving further evidence that recipient BKV was replaced by donor BKV after Tx. CONCLUSIONS: Our study for the first time provides evidence of BKV donor origin in renal transplant recipients.

Factors influencing viral clearing and renal function during polyomavirus BK-associated nephropathy after renal transplantation.

BACKGROUND: The course of BK virus nephropathy (BKVN) is difficult to predict. METHODS: Between 2008 and 2010, we diagnosed BKVN in 46 (5.5%) of 859 patients with transplant biopsies by simian virus 40 (SV40) staining and routine serum polymerase chain reaction. We measured the influence of different variables on glomerular filtration rate (ΔGFR increasing or decreasing) and the time for viral polymerase chain reaction reduction by 1 log (≤13 or >13 weeks). At diagnosis, we either reduced calcineurin inhibitor (CNI) and mycophenolate mofetil by 30% to 50% (n=23), or we switched from CNI to mammalian target of rapamycin (mTOR) inhibitor (n=7) or from CNI to mTOR inhibitor as a second step in patients with protracted viral reduction (n=16). Results are the following: GFR stabilized or increased in 61% of patients and decreased in 39% (graft failure, 15%). Viral reduction by 1 log was rapid in 54% (≤13 weeks) and slow in 46% (>13 weeks). Rapid viral reduction was associated with stable or increasing GFR (84%), compared with slow viral reduction (33%; P=0.0004). High peak viral load, tacrolimus treatment, and late diagnosis (biopsy for cause vs. protocol biopsy) had a negative influence on GFR and viral reduction time. Defining 1-log viral load reduction as an event, tacrolimus compared with cyclosporine was associated with slow viral reduction (P=0.0043). In 88% of patients with slow viral reduction, the secondary switch from CNI to mTOR inhibitor favored viral load decrease. CONCLUSIONS: We conclude that peak viral load, tacrolimus treatment, delayed diagnosis, and viral reduction time influence outcomes in patients with BKVN.