Publications by authors named "Gianluigi Ardissino"

82 Publications

Haemoglobinuria for the early identification of aHUS relapse: data from the ItalKId-HUS Network.

J Nephrol 2021 Jan 18. Epub 2021 Jan 18.

Dialysis and Transplant Unit, Center for HUS Prevention Control and Management at the Pediatric Nephrology, Fondazione IRCCS Ca' Granda-Ospedale Maggiore Policlinico, Via della Commenda 9, 20122, Milan, Italy.

Background: Atypical haemolytic uremic syndrome (aHUS) is at high risk of relapse at any time, therefore patients require lifelong monitoring. The most appropriate way to monitor patients is not yet clear. Patients could be monitored for relapse by urine dipstick testing for haemoglobinuria based on the hypothesis that thrombotic microangiopathy involving the glomerulus and associated with renal damage (like aHUS) cannot occur without haematuria.

Methods: The aim of this retrospective study is to analyse our experience with this approach in aHUS patients who have never previously been treated, who are currently on treatment or who have discontinued C5 inhibition. The records of all aHUS patients (children and adults) managed by or referred to our Centre from January 2009 to March 2020 were included and the analysis for the presence of haemoglobinuria was restricted to the period following primary remission. A positive test was defined as haemoglobin ≥ 1 + . Patients reporting positive urine dipstick tests underwent laboratory investigations to rule in or out the diagnosis of aHUS relapse.

Results: Eighty-four patients were included with 1517 determinations of haemoglobinuria during a cumulative observation period of 8904 patient-months. Haemoglobinuria for the early diagnosis of ongoing aHUS relapse shows a sensitivity of 100% and a specificity of 87.4% with a positive predictive value (PPV) of 10.5% and a negative predictive value (NPV) of 100%. The accuracy of the test was 87.6%.

Conclusion: Haemoglobinuria is a very sensitive and acceptably specific marker of aHUS relapse. This finding and its validation may have a positive impact on patients' quality of life and on the outcome of this life threatening disease via early diagnosis of relapse.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s40620-021-00965-8DOI Listing
January 2021

Family Clusters of Shiga Toxin-producing Escherichia coli Infection: An Overlooked Source of Transmission. Data From the ItalKid-Hus Network.

Pediatr Infect Dis J 2021 Jan;40(1):1-5

Center for HUS Prevention Control and Management, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.

Background: The aim of the present work was to investigate family clusters of Shiga toxin-producing Escherichia coli (STEC) infection among the household members of STEC positive patients, identified within a screening program of bloody diarrhea (BD) for STEC in Northern Italy.

Methods: Stool samples from patients with BD or BD-associated-hemolytic uremic syndrome (HUS) and related households were investigated by molecular and bacteriologic methods to detect and characterize the virulence profile of STEC and Pulsed Field Gel Electrophoresis analysis were done on isolates.

Results: Thirty-nine cases of STEC infection (isolated BD in 16, BD-associated-HUS in 23) were considered, and a total of 130 stool samples from 1 to 8 households of the index patient were analyzed. The prevalence of positivity was higher in siblings (34.8%, 8/23) than in mothers (20%, 7/35), grandparents (9.5%, 2/21), fathers (8.8%, 3/34) or other households. In 14 clusters (36%), one or more household shared a STEC with the same virulence profile (stx, eae, serogroup) as the index case. In 7 clusters, STEC strains isolated from at least 2 subjects also shared identical Pulsed Field Gel Electrophoresis profile. The frequency of household infection does not appear to be associated to the index case's illness (HUS or BD), nor with the serotype or with the virulence profile of the involved STEC (stx2 or stx1-stx2).

Conclusions: Our study shows that STEC infections, most likely related to human-to-human transmission, are common among households of patients with STEC BD or HUS and underlines the importance of extending the epidemiologic investigations to all family members, as the index case may not always be the primary infection in the family.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/INF.0000000000002877DOI Listing
January 2021

Genetic and molecular evidence for complement dysregulation in patients with HELLP syndrome.

Thromb Res 2020 12 27;196:167-174. Epub 2020 Aug 27.

Internal Medicine, Department of Pathophysiology and Transplantation, University of Milan, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, Via Pace 9, 20122 Milan, Italy. Electronic address:

Background: HELLP (Hemolysis, elevated liver enzymes and low platelets) syndrome is a severe and acute pregnancy-related disorder that occurs in approximately 2.5 per 1000 deliveries and represents a major cause of maternal and perinatal morbidity and mortality. This syndrome has been suggested to be a microangiopathy and delivery is the only effective treatment.

Objectives: The aim of this study was to investigate the pathophysiology of HELLP syndrome by simultaneously exploring complement, haemostasis, autoimmunity and inflammation in relation to the clinical outcome.

Methods: We investigated 19 HELLP patients at the time of diagnosis and 3 months after delivery, for complement function, haemostasis and inflammation with immunoenzymatic methods. Complement-related gene variants were also analyzed by next generation sequencing and multiplex ligation-dependent probe amplification. Nineteen age-matched healthy pregnant women served as controls.

Results: At diagnosis, HELLP patients, compared to controls, showed significantly higher plasma levels of SC5b-9 (median 710 ng/ml [range 216-1499] vs 253 ng/ml [19-371], P < 0.0001) and of C5a (20.8 ng/ml [5.6-27.5] vs 12.7 ng/ml [3.2-24.6]; P = 0.004), which decreased three months after delivery (SC5b9: 190 ng/ml [83-446] vs 160 ng/ml [107-219]; C5a: 9.28 ng/ml [2.3-21.6] vs 10.7 ng/ml [2.5-21.2]). A significantly higher frequency of genetic variants involving complement regulatory genes was also observed (52.6% vs 15.8%; P = 0.016). Moreover, at HELLP diagnosis, patients showed increased coagulation markers (fragment F1 + 2 and D-dimer; P = 0.0001) while both patients and controls had high thrombin-generation potential that decreased after delivery.

Conclusions: In the pathophysiology of HELLP syndrome, complement dysregulation, in addition to coagulation activation, is involved and may represent a potential target for treatment with the aim of delaying delivery.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.thromres.2020.08.038DOI Listing
December 2020

Is Shigatoxin 1 protective for the development of Shigatoxin 2-related hemolytic uremic syndrome in children? Data from the ItalKid-HUS Network.

Pediatr Nephrol 2020 10 30;35(10):1997-2001. Epub 2020 Jul 30.

Center for HUS Prevention, Control and Management, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, via Commenda 9, 20122, Milan, Italy.

Background: Shigatoxin (Stx)-producing Escherichia coli (STEC) are the most common causes of hemolytic uremic syndrome (STEC-HUS). The aim of our study is to compare the risk of developing STEC-HUS in relation to the type of Stx genes (Stx1, Stx2, or both).

Methods: This is a prospective, observational, multicenter study involving 63 pediatric units in Northern Italy (ItalKid-HUS Network). STEC-infected children were identified within a screening program for bloody diarrhea during a 10-year period (2010-2019). Stx genes were detected by reverse dot blot or real-time PCR. After the identification of STEC infection, children were followed until diarrhea complete recovery for the possible development of STEC-HUS.

Results: Of the 214 Stx-positive patients, 34 (15.9%) developed STEC-HUS. The risk of HUS in STEC-infected children with Stx1 (n: 62; 29.0%) and Stx2 (n: 97; 45.3%) was respectively 0% and 23.7%, while in patients carrying both Stx1 and Stx2 (n: 55; 25.7%), the risk was 12.7% (p: 0.001).

Conclusions: Our data confirm that Stx1 is a very rare cause of STEC-HUS and demonstrate that the risk of STEC-HUS halves in the case of Stx1+2-producing Escherichia coli infection compared with infections where Stx2 is present alone. This observation is helpful in assessing the risk of individual STEC-infected patients for the development of HUS and suggests that Stx1, in the presence of Stx2, might exert a protective role possibly by receptor competition.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00467-020-04697-yDOI Listing
October 2020

Multiple office blood pressure measurement: a novel approach to overcome the weak cornerstone of blood pressure measurement in children. Data from the SPA project.

Pediatr Nephrol 2020 04 3;35(4):687-693. Epub 2020 Jan 3.

Pediatric Nephrology, Dialysis and Transplantation Unit - Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.

Background: This contribution aims to report and analyze a novel approach for office blood pressure measurement in children.

Methods: Healthy children 5 to 8 years of age were eligible. After 5 minutes rest, 10 unattended blood pressure readings were taken at 3-minute intervals using a validated automated oscillometric device. After discarding outlier values (< 5th or > 95th percentile of the recorded values), the coefficient of variation and the mean of the 10 readings were calculated. The single readings #1 to #10 were compared with this elaborated average of the 10 measurements.

Results: Two hundred eighty-one healthy, non-obese children (137 females, 49%), median age 5.7 (IQR 5.3-6.1) years, were analyzed. The median coefficients of variation were 7% (IQR 5-9) for systolic and 4% (IQR 3-6) for diastolic blood pressure. The first 3 measurements were significantly different from the average, while the readings #4 to #10 were not. Based on the average, only nine subjects had a systolic or diastolic blood pressure > 90th centile (n = 3 > 95th percentile).

Conclusions: Although most guidelines advise three blood pressure readings, these findings suggest that in children, office blood pressure measurement might be improved by including ten measurements. In situations of time constraints, the fourth blood pressure reading might be used as a reliable approximation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00467-019-04368-7DOI Listing
April 2020

Particulate Shiga Toxin 2 in Blood is Associated to the Development of Hemolytic Uremic Syndrome in Children.

Thromb Haemost 2020 Jan 13;120(1):107-120. Epub 2019 Dec 13.

Center for HUS Prevention Control and Management, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.

Hemolytic uremic syndrome (HUS), the leading cause of acute renal failure in children (< 3 years), is mainly related to Shiga toxins (Stx)-producing (STEC) infections. STEC are confined to the gut resulting in hemorrhagic colitis, whereas Stx are delivered in blood to target kidney and brain, with unclear mechanisms, triggering HUS in 5 to 15% of infected children. Stx were found on circulating cells, free in sera (soluble Stx) or in blood cell-derived microvesicles (particulate Stx), whereby the relationship between these forms of circulating toxins is unclear. Here, we have examined 2,846 children with bloody diarrhea and found evidence of STEC infection in 5%. Twenty patients were enrolled to study the natural course of STEC infections before the onset of HUS. In patients, Stx were found to be associated to circulating cells and/or free and functionally active in sera. In most children, Stx were bound to neutrophils when high amounts of toxins were found in feces. Time-course analysis showed that Stx increased transiently in patients' sera while the decrease of toxin amount on leukocytes was observed. Notably, patients who recovered (85%) displayed different settings than those who developed HUS (15%). The distinctive feature of the latter group was the presence in blood of particulate Stx2 (Stx2 sedimented at -forces corresponding to 1 μm microvesicles) the day before diagnosis of HUS, during the release phase of toxins from circulating cells. This observation strongly suggests the involvement of blood cell-derived particulate Stx2 in the transition from hemorrhagic colitis to HUS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1055/s-0039-3400301DOI Listing
January 2020

[HELLP syndrome and hemolytic uremic syndrome during pregnancy: two disease entities, same causation. Case report and literature review].

G Ital Nefrol 2019 Apr;36(2)

Centro per la Cura e lo Studio della Sindrome Emolitico-Uremica. Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milano.

Thrombotic microangiopathies (TMA) are a group of diseases that can complicate pregnancy and threaten the lives of both the mother and the fetus. Several conditions can lead to TMA, including thrombotic thrombocytopenic purpura (TTP), HELLP syndrome and hemolytic uremic syndrome (HUS). We describe the case of a 39-year-old woman who presented a HELLP syndrome in the immediate postpartum period. The patient had acute kidney injury (AKI), increased LDH, unmeasurable haptoglobin levels and hypocomplementemia. Her ADAMTS13 value was normal, thus ruling out TTP. Shiga toxin tests were negative, so HUS associated with E. coli was also ruled out. HELLP syndrome and atypical hemolytic-uremic syndrome (aHUS) remained the most probable diagnosis. In the days following childbirth, the patient's transaminase and bilirubin levels normalized while the anemia persisted, as did the AKI, resulting in the institution of dialysis treatment. A diagnosis of aHUS was made and therapy with eculizumab was started. The patient's blood counts progressively improved, urine output was restored, her indices of renal function also concomitantly improved and dialysis was interrupted. A rash appeared after the third administration of eculizumab and the treatment was suspended. The patient is currently being followed up and has not relapsed. At thirteen months after delivery her renal function is normal as are her platelet counts, LDH, haptoglobin levels and proteinuria. Tests for mutations in the genes that regulate complement activity were negative. We believe that childbirth triggered the HELLP syndrome, which in turn brought about and sustained the HUS. In fact, the patient's liver function improved right after delivery, while her kidney injury and hemolysis persisted, and she also had an excellent response to eculizumab. To our knowledge, no other cases of HELLP syndrome associated with haemolytic uremic syndrome during pregnancy have been reported in literature, nor have cases in which treatment with eculizumab was limited to only three administrations.
View Article and Find Full Text PDF

Download full-text PDF

Source
April 2019

Haploidentical Hematopoietic Stem Cell Transplant Complicated by Atypical Hemolytic Uremic Syndrome and Kidney Transplant From the Same Donor With No Immunosuppression but C5 Inhibition.

Transplantation 2019 02;103(2):e48-e51

Hematology and Bone Marrow Transplantation Unit, IRCCS S. Raffaele Scientific Institution, Milan, Italy.

Background: Atypical hemolytic uremic syndrome (aHUS) is life-threatening condition particularly when complicating allograft hematopoietic stem cell transplant (HSCT). In the past, the outcome was very poor with the majority of patients reaching end-stage renal disease or dying with little or no chances of kidney transplant (KTx) due to the high risk of relapse. The availability of C5 inhibition has opened up significant therapeutic opportunities and has improved the outcome particularly if complement dysregulation (CD) is the underlying pathogenetic mechanism.

Methods: We describe a peculiar case of a girl with aHUS complicating HSCT and her subsequent successful KTx received from the same donor thus performed without immunosuppression but anti-C5 inhibition.

Results: Soon after HSCT performed for acute lymphoblastic leukemia, the patient developed a TMA due to CD and reached end-stage renal disease. After 2 years on dialysis, the patient received a KTx from her father who was already the HSCT donor. Given the full chimerism, no immunosuppressive agent was prescribed except a short (2 days) course of steroids and eculizumab to prevent aHUS relapse. Nine months after the KTx, the patient is well with normal renal function, no immunosuppression and continues eculizumab prevention of aHUS (1 infusion every 21 days).

Conclusions: All patients with transplant-associated thrombotic microangiopathy should be screened for the causes of CD. C5 inhibition with eculizumab is an important therapeutic resource to manage this complication. When KTx is necessary, immunosuppression can be safely withhold in case of same donor for both grafts and documented full chimerism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/TP.0000000000002505DOI Listing
February 2019

Soluble Toll-Like Receptor 4 Impairs the Interaction of Shiga Toxin 2a with Human Serum Amyloid P Component.

Toxins (Basel) 2018 09 18;10(9). Epub 2018 Sep 18.

Western Regional Research Center, U.S. Department of Agriculture, Agricultural Research Service, 800 Buchanan Street, Albany, CA 94710, USA.

Shiga toxin 2a (Stx2a) is the main virulence factor produced by pathogenic strains (Stx-producing , STEC) responsible for hemorrhagic colitis and the life-threatening sequela hemolytic uremic syndrome in children. The toxin released in the intestine by STEC targets the globotriaosylceramide receptor (Gb3Cer) present on the endothelial cells of the brain and the kidney after a transient blood phase during which Stx2a interacts with blood components, such as neutrophils, which, conversely, recognize Stx through Toll-like receptor 4 (TLR4). Among non-cellular blood constituents, human amyloid P component (HuSAP) is considered a negative modulating factor that specifically binds Stx2a and impairs its toxic action. Here, we show that the soluble extracellular domain of TLR4 inhibits the binding of Stx2a to neutrophils, assessed by indirect flow cytometric analysis. Moreover, by using human sensitive Gb3Cer-expressing cells (Raji cells) we found that the complex Stx2a/soluble TLR4 escaped from capture by HuSAP allowing the toxin to target and damage human cells, as assayed by measuring translation inhibition, the typical Stx-induced functional impairment. Thus, soluble TLR4 stood out as a positive modulating factor for Stx2a. In the paper, these findings have been discussed in the context of the pathogenesis of hemolytic uremic syndrome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/toxins10090379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6162853PMC
September 2018

A simple prognostic index for Shigatoxin-related hemolytic uremic syndrome at onset: data from the ItalKid-HUS network.

Eur J Pediatr 2018 Nov 10;177(11):1667-1674. Epub 2018 Aug 10.

Center for HUS Prevention Control and Management at the Epidemiology Unit, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, Milano, Italy.

Shigatoxin Escherichia coli-related hemolytic uremic syndrome (eHUS) is a severe thrombotic microangiopathy (TMA) burdened by life-threatening complications and long-term sequelae. Since hemoconcentration is associated with worse outcome, we tried to develop a reliable and easy-to-calculate index for predicting complications and sequelae based on hemoglobin (Hb) at presentation. The first laboratory examinations with signs of TMA in eHUS patients were analyzed in relation to the outcomes with the receiver operating characteristic curves and their areas under the curve (AUC) for Hb and creatinine (sCr). A total of 197 eHUS patients were identified of whom 24% did not have anemia at presentation. Hb level was the best predictor of a poor outcome (AUC 0.67) but the combination of Hb with sCr, in the formula [(Hb in g/dL + (sCr in mg/dL × 2)], showed an even better AUC of 0.75. The described scoring system was also strongly associated and predictive of all complications and health care needs (8% of patients with scoring > 13 died or entered a permanent vegetative state compared with 0% of those with ≤ 13).Conclusion: The presented score is a simple and early predictor of both short- and long-term outcomes and identifies patients who should undergo rapid volume expansion to counteract hemoconcentration, the spreading of microvascular thrombosis, and the consequent increased organ damage. What is Known: • In eHUS, hemoconcentration is associated with worse short- and long-term outcome. • A prognostic index to identify patients at higher risk for complications at presentation is not available. What is New: • We developed a simple and early prognostic index for eHUS outcome with the combination of Hb and sCr at onset, in the following formula [(Hb in g/dL + (sCr in mg/dL × 2)]. • The proposed HUS Severity Score can promptly identify patients with good outcome and those with high risk of worse short- and long-term outcome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00431-018-3198-7DOI Listing
November 2018

Clinical and genetic predictors of atypical hemolytic uremic syndrome phenotype and outcome.

Kidney Int 2018 08 19;94(2):408-418. Epub 2018 Jun 19.

Department of Immunology, Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Paris, France.

Atypical hemolytic uremic syndrome (aHUS) is a rare, genetic, life-threatening disease. The Global aHUS Registry collects real-world data on the natural history of the disease. Here we characterize end-stage renal disease (ESRD)-free survival, the rate of thrombotic microangiopathy, organ involvement and the genetic background of 851 patients in the registry, prior to eculizumab treatment. A sex-specific difference was apparent according to age at initial disease onset as the ratio of males to females was 1.3:1 for childhood presentation and 1:2 for adult presentation. Complement Factor I and Membrane Cofactor Protein mutations were more common in patients with initial presentation as adults and children, respectively. Initial presentation in childhood significantly predicted ESRD risk (adjusted hazard ratio 0.55 [95% confidence interval 0.41-0.73], whereas sex, race, family history of aHUS, and time from initial presentation to diagnosis, did not. Patients with a Complement Factor H mutation had reduced ESRD-free survival, whereas Membrane Cofactor Protein mutation was associated with longer ESRD-free survival. Additionally extrarenal organ manifestations occur in 19%-38% of patients within six months of initial disease presentation (dependent on organ). Thus, our real-world results provide novel insights regarding phenotypic variables and genotypes on the clinical manifestation and progression of aHUS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.kint.2018.02.029DOI Listing
August 2018

Serum creatinine during physiological perinatal dehydration may estimate individual nephron endowment.

Eur J Pediatr 2018 Sep 1;177(9):1383-1388. Epub 2018 Feb 1.

Neonatal Intensive Care Unit, Fondazione IRCCS Cà Granda, Ospedale Maggiore Policlinico, University of Milan, via Commenda 12, 20122, Milan, Italy.

It is well known that the nephron endowment of healthy subjects is highly variable and that individual nephron mass has potentially important implications both in health and disease. However, nephron count is technically impossible in living subjects. Based on the observation of an increase in serum creatinine (sCr) in otherwise healthy newborns with solitary kidney during the physiological perinatal dehydration, we hypothesized that perinatal sCr might be helpful in identifying healthy subjects with a reduced nephron mass. In the framework of a study on blood pressure in babies (NeoNeph), sCr of normal Caucasian neonates was determined 48-96 h after birth and their association with a family history of arterial hypertension (AH) was analyzed. SCr was determined in 182 normal newborns (90 males) at a mean of 61 ± 8 h after birth (range 46-82). Newborns with paternal AH had a higher mean sCr (0.97 + 0.28 mg/dL) then newborns without paternal AH (0.73 + 0.28 mg/dL; p = 0.006). No differences in mean sCr were found in relation with mother or grandparent's history of AH.

Conclusion: The association between parental AH and high sCr during perinatal dehydration supports the hypothesis that the latter is a promising tool for identifying normal subjects with a reduced nephron mass with potential important implications in prevention and in understanding the individual outcome of renal and extrarenal diseases (including AH). What is Known: • Nephron endowment of healthy subjects is highly variable and individual nephron mass has potentially important implications both in health and disease however nephron count is not feasible in living subjects. What is New: • Serum creatinine during perinatal dehydration is a possible biomarker for identifying normal subjects with a reduced nephron mass.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00431-018-3087-0DOI Listing
September 2018

An Improved Method for the Sensitive Detection of Shiga Toxin 2 in Human Serum.

Toxins (Basel) 2018 01 31;10(2). Epub 2018 Jan 31.

Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Via San Giacomo 14, 40126 Bologna, Italy.

Shiga toxins (Stx) released by Stx-producing (STEC) are virulence factors that are most closely 3associated with hemolytic uremic syndrome (HUS), a life-threatening complication of intestinal infections by STEC. Stx have to enter into the circulatory system before they are delivered to target organs and cause damage. The presence of Stx in sera could be a risk indicator for HUS development. However, the detection of Stx, particularly Stx2, has been difficult due to the presence of Stx2-binding components in human serum. Here, we report new ELISA-based methods for the detection of Stx1 and Stx2 in human serum and the effect of guanidinium chloride on enhancing the sensitivity for the detection of Stx2. The recovery rate for Stx2 was 62% when Stx2-spiked serum samples were treated with guanidinium chloride at a concentration of 200 mM, in contrast to 17% without guanidinium chloride treatment. The effectiveness of guanidinium chloride treatment for the detection of Stx2 in human serum was validated using sera from STEC-infected patients. Coimmunoprecipitation results indicated a specific physical interaction between Stx2 and the human serum amyloid P component (HuSAP) in human serum samples. Our in vitro study demonstrated that the inhibition from HuSAP alone for the detection of Stx2 was only 20%, much less than 69.6% from human serum at Stx2 level 10 ng/mL, suggesting that there may be other factors that bind Stx2 in human serum. This study indicates that treatment of serum samples with guanidinium chloride may be useful for the early and sensitive detection of Stx2 in sera of STEC-infected patients, so preventive measures can be adopted in a timely manner.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/toxins10020059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5848160PMC
January 2018

Management and outcomes of childhood Goodpasture's disease.

Pediatr Res 2018 04 10;83(4):813-817. Epub 2018 Jan 10.

Pediatric Department of Southern Switzerland, Bellinzona, Switzerland.

BackgroundIn an attempt to improve knowledge about childhood Goodpasture's disease, we performed a retrospective analysis of patients with Goodpasture's disease from several pediatric nephrology centers.MethodsWe analyzed the responses to 27 questions that elicited information about the following: incidence, demographics, patient history and clinical presentation, diagnostics performed, acute and chronic therapy, course of disease, and outcome.ResultsGoodpasture's disease, which is extremely rare in this age group, may manifest in 2-year-old toddlers and does not typically present with pulmonary findings before puberty. Goodpasture's disease has a poor outcome with more than 50% of patients progressing to end-stage renal disease. No deaths were reported in this cohort, and renal improvement was observed in children with severe biopsy findings who required renal replacement therapy during the acute phase.ConclusionThe present investigation gives detailed information about childhood Goodpasture's disease under real-life conditions and reveals that very few pediatric cases have been reported. Nearly 50% of children progressed to end-stage renal disease. However, long-term outcome in children might be better than in adults. Aggressive immunosuppressive therapy might be necessary for all affected children, even in patients who require renal replacement therapy or have severe biopsy findings.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/pr.2017.315DOI Listing
April 2018

Exome-wide Association Study Identifies GREB1L Mutations in Congenital Kidney Malformations.

Am J Hum Genet 2017 Nov;101(5):789-802

Division of Nephrology, Columbia University, New York, NY 10032, USA.

Renal agenesis and hypodysplasia (RHD) are major causes of pediatric chronic kidney disease and are highly genetically heterogeneous. We conducted whole-exome sequencing in 202 case subjects with RHD and identified diagnostic mutations in genes known to be associated with RHD in 7/202 case subjects. In an additional affected individual with RHD and a congenital heart defect, we found a homozygous loss-of-function (LOF) variant in SLIT3, recapitulating phenotypes reported with Slit3 inactivation in the mouse. To identify genes associated with RHD, we performed an exome-wide association study with 195 unresolved case subjects and 6,905 control subjects. The top signal resided in GREB1L, a gene implicated previously in Hoxb1 and Shha signaling in zebrafish. The significance of the association, which was p = 2.0 × 10 for novel LOF, increased to p = 4.1 × 10 for LOF and deleterious missense variants combined, and augmented further after accounting for segregation and de novo inheritance of rare variants (joint p = 2.3 × 10). Finally, CRISPR/Cas9 disruption or knockdown of greb1l in zebrafish caused specific pronephric defects, which were rescued by wild-type human GREB1L mRNA, but not mRNA containing alleles identified in case subjects. Together, our study provides insight into the genetic landscape of kidney malformations in humans, presents multiple candidates, and identifies SLIT3 and GREB1L as genes implicated in the pathogenesis of RHD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ajhg.2017.09.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5673636PMC
November 2017

Complement functional tests for monitoring eculizumab treatment in patients with atypical hemolytic uremic syndrome: an update.

Pediatr Nephrol 2018 03 18;33(3):457-461. Epub 2017 Oct 18.

Center for HUS Prevention, Control, and Management at Medicina Interna, Dipartimento di Fisiopatologia Medico-Chirurgica e dei Trapianti, Università degli Studi di Milano, Milan, Italy.

Background: Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy (TMA) characterized by platelet consumption, hemolysis, and organ damage. Eculizumab (ECU), a humanized antibody that blocks complement activity, has been successfully used in aHUS, but the best treatment schedule is not yet clear.

Methods: Here, we report our experience with ECU maintenance treatment and the interval between subsequent doses being extended based on global classical complement pathway (CCP) activity aimed at <30% for maintaining aHUS into remission.

Results: We report on 38 patients with aHUS, 13 children, 21 female, with a median age of 25.0 years (range 0.5-60) at disease onset treated with ECU standard schedule for a median of 2.6 months (range 0.4-24.6). Once stable TMA remission was obtained, the interval between ECU doses was extended based on complement function, with a target CCP activity of <30%. With this approach, 22 patients regularly receive ECU infusion every 28 days and 16 every 21. During a median observation period on ECU, an extended interval of 26.9 months (range 0.8-80.9), with a cumulative observation period of 1,208 months, none of the patients relapsed.

Conclusion: Monitoring complement activity allows a safe reduction in the frequency of ECU administration in aHUS while keeping the disease in remission.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00467-017-3813-2DOI Listing
March 2018

Late Onset Cobalamin Disorder and Hemolytic Uremic Syndrome: A Rare Cause of Nephrotic Syndrome.

Case Rep Pediatr 2017 1;2017:2794060. Epub 2017 Aug 1.

Pediatric Unit, Fondazione IRCCS Ca' Granda Osp. Maggiore Policlinico, Milan, Italy.

Hemolytic uremic syndrome (HUS) is an unrare and severe thrombotic microangiopathy (TMA) caused by several pathogenetic mechanisms among which Shiga toxin-producing infections and complement dysregulation are the most common. However, very rarely and particularly in neonates and infants, disorders of cobalamin metabolism (CblC) can present with or be complicated by TMA. Herein we describe a case of atypical HUS (aHUS) related to CblC disease which first presented in a previously healthy boy at age of 13.6 years. The clinical picture was initially dominated by nephrotic range proteinuria and severe hypertension followed by renal failure. The specific treatment with high dose of hydroxycobalamin rapidly obtained the remission of TMA and the complete recovery of renal function. We conclude that plasma homocysteine and methionine determinations together with urine organic acid analysis should be included in the diagnostic work-up of any patient with TMA and/or nephrotic syndrome regardless of age.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1155/2017/2794060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5557262PMC
August 2017

Comment to "Blood urea nitrogen to serum creatinine ratio is an accurate predictor of outcome in diarrhea-associated hemolytic uremic syndrome" by Keenswijk et al. Eur J Pediatr 2017; 176(3): 355-360.

Eur J Pediatr 2018 02 19;177(2):269-270. Epub 2017 Aug 19.

Center for HUS Prevention, Control and Management, Epidemiology Unit, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00431-017-2989-6DOI Listing
February 2018

Response.

Biol Blood Marrow Transplant 2017 11 15;23(11):2014-2015. Epub 2017 Aug 15.

Medicina Interna, Dipartimento di Fisiopatologia Medico Chirurgica, Università degli Studi di Milano, IRCCS Fondazione Ca' Granda, Ospedale Maggiore Policlinico, Milano, Italy.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbmt.2017.07.017DOI Listing
November 2017

[Complement factor B mutation in atypical hemolytic uremic syndrome. Rare cause of rare disease].

G Ital Nefrol 2017 Apr;34(2):74-81

Cattedra di Nefrologia, Dipartimento di Medicina clinica e sperimentale, Università di Messina, Messina, Italia.

Hemolytic uremic syndrome (HUS) is a rare disease characterized by microangiopathic hemolysis, platelet consumption and multiple organ failure with predominant renal involvement. In the most of cases (85-90%), it is associated with enteric infection due to Shiga-toxin or verocytotoxin (STEC-VTEC)-producer Escherichia coli. Rarely, in about 10-15% of cases, HUS develops in the presence of a disorder of alternative complement pathway regulation and it is defined atypical (aHUS). We describe the case of a 65-year-old man who came to our attention with a clinical presentation of aHUS and a clinical course characterized by rapidly progressive acute renal failure (ARF), which required renal replacement treatments, and by a stable clinical picture of hematological impairment as a marker of a non-severe and self-limiting form. The clinical and laboratory course allowed us not to perform specific therapies such as plasma exchange and/or block of the complement with eculizumab. Less than two weeks after hospital admission, there was a gradual recovery of renal function with spontaneous diuresis and hematological remission. Genetic screening has revealed a heterozygous mutation in the complement factor B (CFB) that is not described in the literature and therefore not yet characterized in the genotype/phenotype correlation, also for the extreme rarity of the forms associated with CFB alteration. In conclusion, the presence of a new mutation in the CFB, such as the one described in our case, is probably associated with the development of aHUS but has not led to a poor prognosis, as generally reported in the literature for known variants of the CFB.
View Article and Find Full Text PDF

Download full-text PDF

Source
April 2017

Bioimpedance-based rather than weight-based ultrafiltration prescription for children on maintenance hemodialysis
.

Clin Nephrol 2018 Oct;90(4):286-290

The determination of dry weight (DW) in young children on hemodialysis (HD) remains challenging. Bioimpedance analysis (BIA) is a potentially helpful means of estimating the need for ultrafiltration and monitoring body fluids in patients on renal replacement therapy, but its role has not yet been clearly defined. The aim of this paper is to share our experience of prescribing ultrafiltration on the basis of BIA parameters alone. The body weight (BW), resistance (Rx), and reactance (Xc) of a 3-year-old girl on chronic HD were recorded pre- and post-HD over a period of 16 months. The BIA parameter that best correlated with actual ultrafiltration (the difference between pre- and post-HD BW) was identified, and the equivalence between actual ultrafiltration and changes in Xc was derived to obtain the following equation: 1 ohm of Xc = 27.4 g of ultrafiltration. Finally, during 21 consecutive HD sessions, ultrafiltration was exclusively prescribed on the basis of the derived equation (BIA-based prescription) after having defined a target post-HD Xc of 45 ohm. The BIA-based prescription period was compared with 21 consecutive HD sessions in which ultrafiltration was prescribed using the conventional approach based on BW (BW-based prescription). Comparison of BIA-based and BW-based ultrafiltration prescription showed significantly fewer HD sessions complicated by hypotension (19 vs. 50%) or by the need for reinfusion (5 vs. 50%), and a better overall quality of HD sessions (86 vs. 37%). No difference in blood pressure was observed, and no acute fluid overload event was detected in either period. BIA-based ultrafiltration seems to be safe, feasible, and effective. The described approach can be particularly useful in the case of patients with problems in setting or maintaining the correct DW.
.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.5414/CN109109DOI Listing
October 2018

Acquired Complement Regulatory Gene Mutations and Hematopoietic Stem Cell Transplant-Related Thrombotic Microangiopathy.

Biol Blood Marrow Transplant 2017 Sep 15;23(9):1580-1582. Epub 2017 May 15.

Laboratory of Medical Genetics, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milano, Italy.

Hematopoietic stem cell transplant-related thrombotic microangiopathy (HSCT-TMA) is a severe complication whose pathophysiology is unknown. We describe 6 patients in which the disease was associated with complement regulatory gene abnormalities received from their respective donors. It is suggested that mutated and transplanted monocyte-derived cells are responsible for production of abnormal proteins, complement dysregulation, and, ultimately, for the disease. This observation might have important drawbacks as far as HSCT-TMA pathophysiology and treatment are concerned.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbmt.2017.05.013DOI Listing
September 2017

FilmArray™ GI panel performance for the diagnosis of acute gastroenteritis or hemorragic diarrhea.

BMC Microbiol 2017 05 12;17(1):111. Epub 2017 May 12.

Microbiology and Virology Department, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy.

Background: Acute gastroenteritis is a common cause of morbidity and mortality in humans worldwide. The rapid and specific identification of infectious agents is crucial for correct patient management. However, diagnosis of acute gastroenteritis is usually performed with diagnostic panels that include only a few pathogens. In the present bicentric study, the diagnostic value of FilmArray™ GI panels was assessed in unformed stool samples of patients with acute gastroenteritis and in a series of samples collected from pediatric patients with heamorragic diarrhea. The clinical performance of the FilmArray™ gastrointestinal (GI) panel was assessed in 168 stool samples collected from patients with either acute gastroenteritis or hemorragic diarrhea. Samples showing discordant results between FilmArray and routine methods were further analyzed with an additional assay.

Results: Overall, the FilmArray™ GI panel detected at least one potential pathogen in 92/168 (54.8%) specimens. In 66/92 (71.8%) samples, only one pathogen was detected, while in 26/92 (28.2%) multiple pathogens were detected. The most frequent pathogens were rotavirus 13.9% (22/168), Campylobacter 10.7% (18/168), Clostridium difficile 9.5% (16/168), and norovirus 8.9% (15/168). Clostridium difficile was identified only in patients with acute gastroenteritis (p < 0.01), while STEC was detected exclusively in patients with hemorragic diarrhea (p < 0.01). In addition, Campylobacter spp., Salmonella spp., EPEC and E. coli producing Shiga-like toxin were more frequently detected in patients with hemorragic diarrhea (p < 0.05). The overall percent agreement calculated in samples was 73.8% and 65.5%, while 34.5% were discordant. After additional confirmatory analyses, the proportion of discordant samples decreased to 7.7%. Rotavirus and astrovirus were the most frequently unconfirmed pathogens.

Conclusion: In conclusion, the FilmArray™ GI panel has proved to be a valuable new diagnostic tool for improving the diagnostic efficiency of GI pathogens.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12866-017-1018-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5427568PMC
May 2017

Role of climate in the spread of shiga toxin-producing Escherichia coli infection among children.

Int J Biometeorol 2017 Sep 7;61(9):1647-1655. Epub 2017 Apr 7.

Center for Prevention, Control and Management of Hemolytic Uremic Syndrome at the Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.

Haemolytic-uraemic syndrome (HUS) is a rare disease mainly affecting children that develops as a complication of shiga toxin-producing Escherichia coli (STEC) infection. It is characterised by acute kidney injury, platelet consumption and mechanical destruction of red blood cells (haemolysis). In order to test the working hypothesis that the spread of the infection is influenced by specific climatic conditions, we analysed all of the identified cases of infection occurring between June 2010 and December 2013 in four provinces of Lombardy, Italy (Milano, Monza Brianza, Varese and Brescia), in which a STEC surveillance system has been developed as part of a preventive programme. In the selected provinces, we recorded in few days a great number of cases and clusters which are unrelated for spatially distant or for the disease are caused by different STEC serotypes. In order to investigate a common factor that favoured the onset of infection, we have analysed in detail the weather conditions of the areas. The daily series of temperature, rain and relative humidity were studied to show the common climate peculiarities whilst the correlation coefficient and the principal component analysis (PCA) were used to point out the meteorological variable, maximum temperature, as the principal climate element in the onset of the infection. The use of distributed lag non-linear models (DLNM) and the climate indices characterising heat waves (HWs) has allowed to identify the weather conditions associated with STEC infection. The study highlighted a close temporal correlation between STEC infection in children and the number, duration and frequency of heat waves. In particular, if the maximum temperature is greater than 90th percentile, days classified as very hot, for 3 or more consecutive days, the risk of infection is increasing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00484-017-1344-yDOI Listing
September 2017

[Efficacy of eculizumab in a case of pregnancy-associated aHUS].

G Ital Nefrol 2016 Jul-Aug;33(4)

Pregnancy-associated thrombotic microangiopathy (TMA) is a rare condition, but it is burdened by a significant perinatal and maternal morbidity as well as mortality. We describe the case of a 33-year-old woman, who developed a TMA at the 36th week of gestation characterized by increased LDH, haptoglobin consumption, schistocytes, thrombocytopenia and acute renal failure requiring dialysis. There were not gestational hypertension nor proteinuria until the day of hospitalization. ADAMTS 13 deficiency was ruled out and the patient did not have diarrhea. She was initially treated with caesarean section, plasma infusion and plasmapheresis with no benefit. Five days after the onset of TMA, a temptative diagnosis of atypical uremic syndrome (aHUS) was made and the patient was switched to eculizumab. Antibiotic prophylaxis and anti-meningococcal A,B, C, W135 and Y vaccination was performed. TMA rapidly resolved and renal function completely recovered. The newborn had a normal perinatal course. A complement dysregulation was ruled out by testing for mutations on CFH, CFHR3-R1, CFI, MCP, CFB, C3 and for anti CFH antibodies. In conclusion the differential diagnosis of aHUS with HELLP syndrome is often not straightforward. The severity and persistence of TMA, the high mortality associated to peripartum TMA and the risk for irreversible kidney failure require an early therapeutic decision as to the use of eculizumab.
View Article and Find Full Text PDF

Download full-text PDF

Source
October 2017

Mutational Spectrum of CYP24A1 Gene in a Cohort of Italian Patients with Idiopathic Infantile Hypercalcemia.

Nephron 2016 9;133(3):193-204. Epub 2016 Jul 9.

Nephrology, Dialysis and Transplantation Unit, Department of Medical and Surgical Sciences, University of Foggia, Foggia, Italy.

Background/aims: Loss-of-function mutations in the CYP24A1 gene, which encodes the vitamin D-24 hydroxylase, have been recognized as a cause of elevated 1,25-dihydroxyvitamin D concentrations, hypercalcemia, hypercalciuria, nephrocalcinosis and nephrolithiasis in infants and adults. As only a case report describing 2 adult patients has been reported in Italian population, we report here the mutation analysis of CYP24A1 gene in an Italian cohort of 12 pediatric and adult patients with idiopathic infantile hypercalcemia (IIH).

Methods: We performed mutational screening of CYP24A1 gene in a cohort of 12 Italian patients: 8 children with nephrocalcinosis, hypercalcemia and PTH levels <10 pg/ml and 4 adult patients with nephrolithiasis, mild hypercalcemia and PTH levels <10 pg/ml from 11 unrelated Italian families. Clinical and biochemical data were collected. Genomic DNA was extracted from peripheral blood leucocytes using standard methods, and whole coding sequence of CYP24A1 gene was analysed in all patients and family members by polymerase chain reaction and direct sequencing. The potential pathogenicity of the newly identified missense mutations was evaluated by 3 different in silico approaches (Sorting Intolerant from Tolerant, Polyphen and Mutation Taster) and by comparative analysis in 14 different species using ClustalW software.

Results: CYP24A1 bi-allelic mutations were found in 8 individuals from 7 Italian families (7/11; 64%). Overall, 6 different CYP24A1 mutations, including one small deletion (p.Glu143del), 4 missense mutations (p.Leu148Pro; p.Arg396Trp; p.Pro503Leu; p.Glu383Gln) and one nonsense mutation (p.Tyr220*) were identified. Two out of 6 mutations (p.Tyr220* and p.Pro503Leu) were not previously described. Moreover, a new CYP24A1 variant was identified by genetic screening of asymptomatic controls.

Conclusion: To the best of our knowledge, this is the first report of a CYP24A1 molecular analysis performed in an Italian cohort of adult and pediatric Italian patients. This study (1) confirms that CYP24A1 plays a causal role in some but not all cases of IIH (64%); (2) expands the spectrum of known CYP24A1 pathogenic mutations; (3) describes 2 hotspots detected in 50% of all Italian cases; and (4) emphasizes the importance of recognition and genetic diagnosis of CYP24A1 defects in infantile as well as adult hypercalcemia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1159/000446663DOI Listing
February 2017

Improved renal recovery in patients with atypical hemolytic uremic syndrome following rapid initiation of eculizumab treatment.

J Nephrol 2017 Feb 19;30(1):127-134. Epub 2016 Mar 19.

Medical School Hannover, Hannover, Germany.

Background: Eculizumab is approved for atypical hemolytic uremic syndrome (aHUS). Guidelines discuss the importance of prompt treatment. We report a post hoc analysis investigating the effect of baseline factors, including patient characteristics and time from the latest aHUS manifestation to eculizumab initiation, on change from baseline in estimated glomerular filtration rate (eGFR) and other outcomes.

Methods: Data were pooled from four phase 2, open-label, single-arm, prospective clinical studies of eculizumab for patients with aHUS. Multivariate regressions identified predictors of eGFR change from baseline. The proportion of patients achieving sustained eGFR increase (defined: ≥15 ml/min/1.73 m for ≥28 days) and platelet count normalization were evaluated 1 year post-treatment. Baseline characteristics and eGFR outcomes were summarized by time to treatment from last aHUS manifestation [≤7 days (n = 21) versus >7 days (n = 76)].

Results: Baseline eGFR were similar between groups. Multivariate regression analysis demonstrated time from aHUS manifestation to eculizumab treatment, age, baseline lactate dehydrogenase (LDH) and baseline hemoglobin were independently predictive of eGFR change from baseline. Mean eGFR change from baseline at 1 year was significantly higher in patients treated in ≤7 days than >7 days (57 vs. 23 ml/min/1.73 m, p = 0.0098). After 1 year, 17/21 and 36/76 patients in the ≤7 and >7 day groups, respectively, achieved a sustained increase in eGFR. Mean time to platelet count normalization was similar between groups.

Conclusions: Younger age, higher baseline LDH and lower baseline hemoglobin were associated with greater eGFR improvements. Early eculizumab initiation led to improved renal recovery, demonstrating the importance of rapid diagnosis and treatment of patients with aHUS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s40620-016-0288-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5316393PMC
February 2017

Eculizumab is a safe and effective treatment in pediatric patients with atypical hemolytic uremic syndrome.

Kidney Int 2016 Mar 28;89(3):701-11. Epub 2016 Jan 28.

The Hospital for Sick Children, Toronto, Ontario, Canada.

Atypical hemolytic uremic syndrome (aHUS) is caused by alternative complement pathway dysregulation, leading to systemic thrombotic microangiopathy (TMA) and severe end-organ damage. Based on 2 prospective studies in mostly adults and retrospective data in children, eculizumab, a terminal complement inhibitor, is approved for aHUS treatment. Here we prospectively evaluated efficacy and safety of weight-based dosing of eculizumab in eligible pediatric patients with aHUS in an open-label phase II study. The primary end point was complete TMA response by 26 weeks. Twenty-two patients (aged 5 months-17 years) were treated; 16 were newly diagnosed, 12 had no prior plasma exchange/infusion during current TMA symptomatology, 11 received baseline dialysis and 2 had prior renal transplants. By week 26, 14 achieved a complete TMA response, 18 achieved hematologic normalization, and 16 had 25% or better improvement in serum creatinine. Plasma exchange/infusion was discontinued in all, and 9 of the 11 patients who required dialysis at baseline discontinued, whereas none initiated new dialysis. Eculizumab was well tolerated; no deaths or meningococcal infections occurred. Bone marrow failure, wrist fracture, and acute respiratory failure were reported as unrelated severe adverse events. Thus, our findings establish the efficacy and safety of eculizumab for pediatric patients with aHUS and are consistent with proposed immediate eculizumab initiation following diagnosis in children.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.kint.2015.11.026DOI Listing
March 2016