Urinary miR-155-5p and CXCL10 as prognostic and predictive biomarkers of rejection, graft outcome and treatment response in kidney transplantation.

AIMS: MicroRNAs (miRNAs) may be useful biomarkers of rejection and allograft outcome in kidney transplantation. Elevated urinary CXCL10 levels have been associated with acute rejection (AR) and may predict allograft failure. We examined the correlation of miRNA, CXCL10 levels and immunosuppressive drug exposure with AR and graft function in kidney transplant recipients. METHODS: Eighty de novo kidney transplant recipients were recruited from four European centres. All patients received tacrolimus, mycophenolate mofetil, and methylprednisolone. Urinary pellet expression of miR-142-3p, miR-210-3p and miR-155-5p was assessed by quantitative real-time polymerase chain reaction and urinary CXCL10 levels by enzyme-linked immunosorbent assay at the 1st week and the 1st , 2nd , 3rd and 6th months post-transplantation. RESULTS: Eight patients experienced AR. Before and during AR, patients showed a significant increase of urinary miR-142-3p, miR-155-5p and CXCL10 levels and a decrease of miR-210-3p levels. Receiver operating characteristic curve analysis showed that miR-155-5p (area under the curve = 0.875; P = 0.046) and CXCL10 (area under the curve = 0.865; P = 0.029) had excellent capacity to discriminate between rejectors and nonrejectors. The optimal cut-off values for the prognosis of AR were 0.51, with 85% sensitivity and 86% specificity for miR-155-5p and 84.73 pg ml-1 , with 84% sensitivity and 80% specificity for CXCL10. miR-155-5p and CXCL10 levels correlated with glomerular filtration rate. Levels of both biomarkers normalized after recovery of graft function. CONCLUSIONS: The regular early post-transplantation monitoring of urinary miR-155-5p and CXCL10 can help in the prognosis of AR and graft dysfunction. Large prospective randomized multicentre trials are warranted to refine our cut-off values and validate the clinical usefulness of these biomarkers.

Target Enzyme Activity and Phosphorylation of Pathway Molecules As Specific Biomarkers in Transplantation.

Therapeutic drug monitoring of immunosuppressants in the clinic is based on the measurement of blood concentration (pharmacokinetics). However, pharmacokinetic monitoring of immunosuppressants does not allow prediction of individual differences in pharmacological effects on immune cells. Pharmacodynamic (PD) monitoring via direct determination of target enzyme activity and phosphorylation of pathway molecules may enhance therapeutic drug monitoring and allow prediction of individual responses to immunosuppressants. This review discusses the clinical relevance of monitoring the activity of inosine-5'-monophosphate dehydrogenase (IMPDH), the target enzyme of mycophenolic acid, and of the phosphorylation of mechanistic target of rapamycin (mTOR) molecules. Significant progress regarding a robust and practicable assay for the determination of IMPDH activity as a specific PD parameter of mycophenolic acid activity has been achieved. The development of a rapid and reliable IMPDH assay system suitable for use in clinical practice was an important step that allowed thorough pharmacokinetics-PD investigations in large numbers of mycophenolic acid-treated patients. A reproducible and validated IMPDH assay was used in a few clinical trials by different research groups and is based on the chromatographic determination of newly generated xanthine monophosphate in mononuclear cell lysates. This assay requires only small volumes of blood and can be reliably used in multicenter trials; however, more clinical data from larger cohorts are needed to determine its clinical utility. Regarding monitoring of mTOR inhibitors (mTORis), the results of the first study that provided data on measurement of mTOR pathway molecules [p70 ribosomal protein S6 kinase (p70S6 kinase) and phosphorylated ribosomal protein S6] suggest that they are suitable targets for individualized PD monitoring of sirolimus and everolimus after organ transplantation. At present, only the phospho-flow phosphorylated ribosomal protein S6 assay has been validated in vitro and evaluated for confounding factors in vivo. The reported specific biomarkers for IMPDH activity and phosphorylation of mTOR molecules must be validated in clinical settings and multicenter studies to prove their clinical validity.

Simultaneous determination of mycophenolate and its metabolite mycophenolate-7-o-glucuronide with an isocratic HPLC-UV-based method in human plasma and stability evaluation.

OBJECTIVES: Mycophenolic acid (MPA) is an immunosuppressive agent which is commonly used in a fixed dose regime in solid organ transplantation. For clinical trials and therapeutic drug monitoring measuring plasma concentrations is necessary. Also, stability issues have to be addressed. METHODS: We describe an isocratic, RP-based HPLC-UV method for simultaneous determination of MPA and its major metabolite Mycophenolic acid 7-o Glucuronide (MPAG) in human plasma. Pre-analytics included protein precipitation with acetonitrile. The method was validated according to EMA/FDA guidelines. Patient lithium-heparin plasma and blood was used for evaluation of short-term (72 hours at room temperature = RT) and long-term stability (2 years at -80 °C) without acidification. RESULTS: Linearity was assessed in the concentration range of 0.5-40.0 µg/mL for MPA and 5.0-350.0 µg/mL for MPAG, respectively. For MPA coefficient of variation was <7.0% (lower limit of quantification = LLOQ: 10.8%), for MPAG <9.6% (LLOQ: 10.6%). Bias ranged between -1.9 and +1.5% for MPA and for MPAG between -4.3 and -0.3%. The method showed agreement with a reference method for both analytes. MPA remained stable for 7 h (-1.6 to +8.4% change to the initial concentration) and MPAG for 24 h (-1.8 to -11.5% change) at RT in lithium heparin blood. After 2 years of storage at -80 °C MPA, MPAG concentrations and 95% CIs remained within ±15% of the initial value. CONCLUSION: The presented assay is applicable for clinical studies. Blood samples were stable for 7 hours at RT and plasma for 2 years stored at -80 °C.

No relevant pharmacokinetic interaction between pantoprazole and mycophenolate in renal transplant patients: a randomized crossover study.

AIMS: Mycophenolic acid (MPA) suppresses lymphocyte proliferation through inosine monophosphate dehydrogenase (IMPDH) inhibition. Two formulations have been approved: mycophenolate mofetil (MMF) and enteric-coated mycophenolate sodium (EC-MPS). Pantoprazole (PAN) inhibits gastric acid secretion, which may alter MPA exposure. Data from healthy volunteers suggest a significant drug-drug interaction (DDA) between pantoprazole and MPA. In transplant patients, a decreased MPA area under the concentration-time curve (AUC) may lead to higher IMPDH activity, which may lead to higher acute rejection risk. Therefore this DDA was evaluated in renal transplant patients under maintenance immunosuppressive therapy. METHODS: In this single-centre, open, randomized, four-sequence, four-treatment crossover study, the influence of PAN 40 mg on MPA pharmacokinetics such as (dose-adjusted) AUC0-12 h (dAUC) was analysed in 20 renal transplant patients (>6 months post-transplantation) receiving MMF (1-2 g day(-1) ) and EC-MPS in combination with ciclosporin. The major metabolite MPA glucuronide (MPAG) and the IMPDH activity were also examined. RESULTS: MMF + PAN intake led to a lowest mean dAUC for MPA of 41.46 ng h ml(-1) mg(-1) [95% confidence interval (CI) 32.38, 50.54], while MPA exposure was highest for EC-MPS + PAN [dAUC: 46.30 ng h ml(-1) mg(-1) (95% CI 37.11, 55.49)]. Differences in dAUC and dose-adjusted maximum concentration (dCmax) were not significant. Only for MMF [dAUC: 41.46 ng h ml(-1) mg(-1) (95% CI 32.38, 50.54)] and EC-MPS [dAUC: 43.39 ng h ml(-1) mg(-1) (95% CI 33.44, 53.34)] bioequivalence was established for dAUC [geometric mean ratio: 101.25% (90% CI 84.60, 121.17)]. Simultaneous EC-MPS + PAN intake led to an earlier time to Cmax (tmax) [median: 2.0 h (min-max: 0.5-10.0)] than EC-MPS intake alone [3 h (1.5-12.0); P = 0.037]. Tmax was not affected for MMF [1.0 h (0.5-5.0)] ± pantoprazole [1.0 h (0.5-6.0), P = 0.928). No impact on MPAG pharmacokinetics or IMPDH activity was found. CONCLUSION: Pantoprazole influences EC-MPS and MMF pharmacokinetics but as it had no impact on MPA pharmacodynamics, the immunosuppressive effect of the drug was not impaired.

[GRADE guidelines 20: Assessing the certainty of evidence in the importance of outcomes or values and preferences - inconsistency, imprecision, and other domains]. / GRADE Leitlinien: 20. Untersuchung der Vertrauenswürdigkeit in die Evidenz zur Beurteilung der Wichtigkeit von Endpunkten oder Werten und Präferenzen ­ Inkonsistenz, unzureichende Präzision und andere Domänen.

OBJECTIVE: To provide Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) guidance for assessing inconsistency, imprecision, and other domains for the certainty of evidence about the relative importance of outcomes. STUDY DESIGN AND SETTING: We applied the GRADE domains to rate the certainty of evidence in the importance of outcomes to several systematic reviews, iteratively reviewed draft guidance, and consulted GRADE members and other stakeholders for feedback. RESULTS: We describe the rationale for considering the remaining GRADE domains when rating the certainty in a body of evidence for the relative importance of outcomes. As meta-analyses are not common in this context, inconsistency and imprecision assessments are challenging. Furthermore, confusion exists about inconsistency, imprecision, and true variability in the relative importance of outcomes. To clarify this issue, we suggest that the true variability is neither equivalent to inconsistency nor imprecision. Specifically, inconsistency arises from population, intervention, comparison and outcome and methodological elements that should be explored and, if possible, explained. The width of the confidence interval and sample size inform judgments about imprecision. We also provide suggestions on how to detect publication bias and discuss the domains to rate up the certainty. CONCLUSION: We provide guidance and examples for rating inconsistency, imprecision, and other domains for a body of evidence describing the relative importance of outcomes.

[GRADE Guidelines: 19. Assessing the certainty of evidence in the importance of outcomes or values and preferences: Risk of bias and indirectness]. / GRADE-Leitlinien: 19. Bewertung der Vertrauenswürdigkeit der Evidenz für die Bedeutung von Endpunkten oder Werten und Präferenzen ­ Risiko für Bias und Indirektheit.

OBJECTIVES: The Grading of Recommendations Assessment, Development, and Evaluation (GRADE) working group defines patient values and preferences as the relative importance patients place on the main health outcomes. We provide GRADE guidance for assessing the risk of bias and indirectness domains for certainty of evidence about the relative importance of outcomes. STUDY DESIGN AND SETTING: We applied the GRADE domains to rate the certainty of evidence in the importance of outcomes to several systematic reviews, iteratively reviewed draft guidance and consulted GRADE members and other stakeholders for feedback. RESULTS: This is the first of two articles. A body of evidence addressing the importance of outcomes starts at "high certainty"; concerns with risk of bias, indirectness, inconsistency, imprecision, and publication bias lead to downgrading to moderate, low, or very low certainty. We propose the following subdomains of risk of bias: selection of the study population, missing data, the type of measurement instrument, and confounding; we have developed items for each subdomain. The population, intervention, comparison, and outcome elements associated with the evidence determine the degree of indirectness. CONCLUSION: This article provides guidance and examples for rating the risk of bias and indirectness for a body of evidence summarizing the importance of outcomes.

Authors should clearly report how they derived the overall rating when applying AMSTAR 2-a cross-sectional study.

OBJECTIVES: A measurement tool to assess systematic reviews (SRs) 2 (AMSTAR 2) allows for deriving the overall confidence in an SR. We investigated how authors derived the overall confidence rating and whether different schemes lead to different results. STUDY DESIGN AND SETTING: We compared three different schemes (original 7-item scheme, a self-developed 5-item scheme, and the AMSTAR Web site) to derive the overall confidence in AMSTAR 2 using two distinct samples of SRs. Multiple bibliographic databases were searched for articles to analyze how AMSTAR 2 was applied by others. RESULTS: In both samples (n = 60 and n = 58), the Friedman test revealed a significant difference between the schemes (P < 0.001). The Web site scheme was the least strict one, whereas between the 5-item and 7-item scheme, no differences were found in post hoc analyses. We included 53 publications applying AMSTAR 2 identified in our literature search. Only 37 of them (70%) used the original 7-item scheme. Less than half of them (18 of 37) reported how they derived the overall rating. CONCLUSION: Authors should clearly report how they have derived the overall rating when applying AMSTAR 2. Reporting should allow for reproducing the overall ratings for editors, peer reviewers, and readers.

The methodological quality of systematic reviews on the treatment of adult major depression needs improvement according to AMSTAR 2: A cross-sectional study.

BACKGROUND: Several standards have been developed to assess methodological quality of systematic reviews (SR). One widely used tool is the AMSTAR. A recent update - AMSTAR 2 - is a 16 item evaluation tool that enables a detailed assessment of SR that include randomised (RCT) or non-randomised studies (NRS) of healthcare interventions. METHODS: A cross-sectional study of SR on pharmacological or psychological interventions in major depression in adults was conducted. SR published during 2012-2017 were sampled from MEDLINE, EMBASE and the Cochrane Database of SR. Methodological quality was assessed using AMSTAR 2. Potential predictive factors associated with quality were examined. RESULTS: In rating overall confidence in the results of 60 SR four reviews were rated "high", two were "moderate", one was "low" and 53 were "critically low". The mean AMSTAR 2 percentage score was 45.3% (standard deviation 22.6%) in a wide range from 7.1% to 93.8%. Predictors of higher quality were: type of review (higher quality in Cochrane Reviews), SR including only randomized trials and higher journal impact factor. LIMITATIONS: AMSTAR 2 is not intended to be used for the generation of a percentage score. CONCLUSIONS: According to AMSTAR 2 the overall methodological quality of SR on the treatment of adult major depression needs improvement. Although there is a high need for summarized information in the field of mental health, this work demonstrates the need to critically assess SR before using their findings. Better adherence to established reporting guidelines for SR is needed.

[GRADE guidelines: 18. How ROBINS-I and other tools to assess risk of bias in nonrandomized studies should be used to rate the certainty of a body of evidence]. / GRADE-Leitlinien: 18. Wie ROBINS-I und andere Instrumente zur Einschätzung des Risikos für Bias von nicht-randomisierten Studien verwendet werden sollten, um die Vertrauenswürdigkeit eines Evidenzkörpers zu bewerten.

OBJECTIVE: To provide guidance on how systematic review authors, guideline developers, and health technology assessment practitioners should approach the use of the risk of bias in nonrandomized studies of interventions (ROBINS-I) tool as a part of GRADE's certainty rating process. STUDY DESIGN AND SETTING: The study design and setting comprised iterative discussions, testing in systematic reviews, and presentation at GRADE working group meetings with feedback from the GRADE working group. RESULTS: We describe where to start the initial assessment of a body of evidence with the use of ROBINS-I and where one would anticipate the final rating would end up. The GRADE accounted for issues that mitigate concerns about confounding and selection bias by introducing the upgrading domains: large effects, dose-effect relations, and when plausible residual confounders or other biases increase certainty. They will need to be considered in an assessment of a body of evidence when using ROBINS-I. CONCLUSION: The use of ROBINS-I in GRADE assessments may allow for a better comparison of evidence from randomized controlled trials (RCTs) and nonrandomized studies (NRSs) because they are placed on a common metric for risk of bias. Challenges remain, including appropriate presentation of evidence from RCTs and NRSs for decision-making and how to optimally integrate RCTs and NRSs in an evidence assessment.

A psychometric study found AMSTAR 2 to be a valid and moderately reliable appraisal tool.

OBJECTIVES: The objectives of this study were to determine the interrater reliability (IRR) of assessment of multiple systematic reviews (AMSTAR) 2 for reviews of pharmacological or psychological interventions for the treatment of major depression, to compare it to that of AMSTAR and risk of bias in systematic reviews (ROBIS), and to assess the convergent validity between the appraisal tools. STUDY DESIGN AND SETTING: Two groups of four raters were each assigned one of two samples of 30 systematic reviews. All eight raters applied AMSTAR 2 to their sample. Each group also applied either AMSTAR or ROBIS. Fleiss' kappa and Gwet's AC1 were calculated, and agreement between the tools was assessed. RESULTS: The median kappa values as a measure of IRR indicated a moderate agreement for AMSTAR 2 (median = 0.51), a substantial agreement for AMSTAR (median = 0.62), and a fair agreement for ROBIS (median = 0.27). Validity results showed a positive association for AMSTAR and AMSTAR 2 (r = 0.91) as well as ROBIS and AMSTAR 2 (r = 0.84). For the overall rating, AMSTAR 2 showed a high concordance with ROBIS and a lower concordance with AMSTAR. CONCLUSION: The IRR of AMSTAR 2 was found to be slightly lower than the IRR of AMSTAR and higher than the IRR of ROBIS. Validity measurements indicate that AMSTAR 2 is closely related to both ROBIS and AMSTAR.

High frequency of valganciclovir underdosing for cytomegalovirus prophylaxis after renal transplantation.

BACKGROUND: The correct valganciclovir dose for cytomegalovirus (CMV) prophylaxis depends on renal function estimated by the Cockcroft-Gault (CG) estimated creatinine clearance (CG-CrCl) formula. Patients with delayed or rapidly changing graft function after transplantation (tx) will need dose adjustments. METHODS: We performed a retrospective investigation of valganciclovir dosing in renal transplant patients receiving CMV prophylaxis between August 2003 and August 2011, and analysed valganciclovir dosing, CG-CrCl, CMV viraemia (CMV-PCR <750 copies/mL), leucopenia (<3500/µL) and neutropenia (<1500/µL) in the first year post-transplant. On Days 30 and 60 post-transplant, dosing pattern in relation to estimated creatinine clearance was analysed regarding CMV viraemia, leucopenia and neutropenia. RESULTS: Six hundred and thirty-five patients received valganciclovir prophylaxis that lasted 129 ± 68 days with a mean dose of 248 ± 152 mg/day of whom 112/635 (17.7%) developed CMV viraemia, 166/635 (26.1%) leucopenia and 48/635 (7.6%) neutropenia. CMV resistance within 1 year post-transplant was detected in three patients. Only 137/609 (22.6%) patients received the recommended dose, while n = 426 (70.3%) were underdosed and n = 43 (7.1%) were overdosed at Day 30 post-tx. Risk factors for CMV viraemia were donor positive D (+)/receptor negative R (-) status and short prophylaxis duration, but not low valganciclovir dose. Risk factors for developing leucopenia were D+/R- status and low renal function. No significant differences in dosing frequency were observed in patients developing neutropenia or not (P = 0.584). CONCLUSION: Most patients do not receive the recommended valganciclovir dose. Despite obvious underdosing in a large proportion of patients, effective prophylaxis was maintained and it was not associated as a risk factor for CMV viraemia or leucopenia.