Serum Neurofilaments are a reliable biomarker to early detect PML in Multiple Sclerosis patients.

BACKGROUND: The earliest detection of progressive multifocal leukoencephalopathy (PML) is crucial in Natalizumab (NTZ)-treated Multiple Sclerosis (MS) patients. This study aims to assess serum Neurofilaments (sNFL) ability to early detect PML in longitudinal patients' follow-up. METHODS: NFL were retrospectively measured in four PML cases occurred at the Regional Referring Center for MS (CRESM, Italy), in samples collected since one year before PML diagnosis, at PML diagnosis, during PML and in post-PML follow-up. sNFL levels were interpreted according to previously defined reference values. Clinical examination and EDSS were performed at each NTZ infusion. Routinary MRI was undertaken every six months; after PML diagnosis, MRI was performed according to clinical evaluation. sNFL were also measured in 45 NTZ-treated patients experiencing NEDA-3 status for at least 12 months. RESULTS: Patients showed different PML onsets and manifestations: in 3 patients routinary brain MRI revealed radiological signs of PML preceding different clinical manifestations, while in one patient brain MRI was performed after the clinical onset. PML diagnosis was defined at the time of the first detection of JCV DNA in cerebrospinal fluid. The following different PML phases were considered: 1. Basal (up to 4 months before PML diagnosis): sNFL values were in the normal range in all patients' samples, except for one (median 9.1 pg/ml, range 6.2-15.1 pg/ml) 2. Pre-PML (within 3 months before PML diagnosis): sNFL were elevated in all available samples (median 19.50 pg/ml, range 15.50-33.80 pg/ml). 3. PML diagnosis: sNFL were elevated in all patients (median 59.20 pg/ml, range 11.1-101.50 pg/ml). 4. PML/IRIS: during this phase, sNFL levels reached their peak (median 96.35 pg/ml, range 20.5-272.9) in all patients. 5. Post-PML (recovery phase, starting from the first MRI without enhancement, up to the end of follow-up): sNFL levels showed a decrease (median 12.80 pg/ml, range 9.30-30.60); however, based on reference values, sNFL were still elevated in 2 out of 4 patients at the end of their follow-up (622 and 887 days after PML diagnosis). sNFL were always elevated when MRI scan suggested a suspicious of PML. In NEDA-3 patients, sNFL levels were in the normal range in all patients' samples (median 4.7 pg/ml, range 1.4-8.6 pg/ml). CONCLUSION: Elevated sNFL were observed not only at PML diagnosis, but also in pre-PML phase. At PML recovery, sNFL weren't normalized in all patients' samples, suggesting ongoing neuronal degeneration. sNFL represent a reliable biomarker and should be introduced in clinical practice as an additional/alternative parameter to MRI to early detect and monitor PML.

The unexplored role of sedentary time and physical activity in glucose and lipid metabolism-related placental mRNAs in pregnant women who are obese: the DALI lifestyle randomised controlled trial.

OBJECTIVE: We aimed to explore: (i) the association of sedentary time (ST) and physical activity (PA) during pregnancy with the placental expression of genes related to glucose and lipid metabolism in pregnant women who are obese; (ii) maternal metabolic factors mediating changes in these placental transcripts; and (iii) cord blood markers related to the mRNAs mediating neonatal adiposity. DESIGN: Multicentre randomised controlled trial. SETTING: Hospitals in nine European countries. POPULATION: A cohort of 112 pregnant women with placental tissue. METHODS: Both ST and moderate-to-vigorous PA (MVPA) levels were measured objectively using accelerometry at three time periods during pregnancy. MAIN OUTCOME MEASURES: Placental mRNAs (FATP2, FATP3, FABP4, GLUT1 and PPAR-γ) were measured with NanoString technology. Maternal and fetal metabolic markers and neonatal adiposity were assessed. RESULTS: Longer periods of ST, especially in early to middle pregnancy, was associated with lower placental FATP2 and FATP3 expression (P < 0.05), whereas MVPA at baseline was inversely associated with GLUT1 mRNA (P = 0.02). Although placental FATP2 and FATP3 expression were regulated by the insulin-glucose axis (P < 0.05), no maternal metabolic marker mediated the association of ST/MVPA with placental mRNAs (P > 0.05). Additionally, placental FATP2 expression was inversely associated with cord blood triglycerides and free fatty acids (FFAs; P < 0.01). No cord blood marker mediated neonatal adiposity except for cord blood leptin, which mediated the effects of PPAR-γ on neonatal sum of skinfolds (P < 0.05). CONCLUSIONS: In early to middle pregnancy, ST is associated with the expression of placental genes linked to lipid transport. PA is hardly related to transporter mRNAs. Strategies aimed at reducing sedentary behaviour during pregnancy could modulate placental gene expression, which may help to prevent unfavourable fetal and maternal pregnancy outcomes. TWEETABLE ABSTRACT: Reducing sedentary behaviour in pregnancy might modulate placental expression of genes related to lipid metabolism in women who are obese.

Metabolic phenotypes of early gestational diabetes mellitus and their association with adverse pregnancy outcomes.

AIMS: To describe the metabolic phenotypes of early gestational diabetes mellitus and their association with adverse pregnancy outcomes. METHODS: We performed a post hoc analysis using data from the Vitamin D And Lifestyle Intervention for gestational diabetes prevention (DALI) trial conducted across nine European countries (2012-2014). In women with a BMI ≥29 kg/m2 , insulin resistance and secretion were estimated from the oral glucose tolerance test values performed before 20 weeks, using homeostatic model assessment of insulin resistance and Stumvoll first-phase indices, respectively. Women with early gestational diabetes, defined by the International Association of Diabetes and Pregnancy Study Groups criteria, were classified into three groups: GDM-R (above-median insulin resistance alone), GDM-S (below-median insulin secretion alone), and GDM-B (combination of both) and the few remaining women were excluded. RESULTS: Compared with women in the normal glucose tolerance group (n = 651), women in the GDM-R group (n = 143) had higher fasting and post-load glucose values and insulin levels, with a greater risk of having large-for-gestational age babies [adjusted odds ratio 3.30 (95% CI 1.50-7.50)] and caesarean section [adjusted odds ratio 2.30 (95% CI 1.20-4.40)]. Women in the GDM-S (n = 37) and GDM-B (n = 56) groups had comparable pregnancy outcomes with those in the normal glucose tolerance group. CONCLUSIONS: In overweight and obese women with early gestational diabetes, higher degree of insulin resistance alone was more likely to be associated with adverse pregnancy outcomes than lower insulin secretion alone or a combination of both.

Postpartum screening for type 2 diabetes mellitus in women with gestational diabetes: Is it really performed?

AIMS: This study evaluates the adherence to postpartum type 2 diabetes mellitus (T2DM) screening in women with previous gestational diabetes (GDM) and identifies elements associated with poor attendance. METHODS: We retrospectively collected data from 650 consecutive women with GDM between 2016 and 2018, who should had 75 g-OGTT, 4-12 weeks after delivery. Impaired glucose regulation (IGR) was defined according with ADA criteria. RESULTS: Only 41% of women had postpartum OGTT. Of these, 1.9% received T2DM diagnosis, with IGR prevalence of 18%. After introducing a recommendation letter, adherence to screening increased (47% in 2017 and 43% in 2018 vs. 32% in 2016). Screening procedure was less common in women with: no-family history of T2DM (38% vs. 46%; p < 0.05), age <35 (33% vs. 47%; p < 0.01), lower level of education (32% no-high-school-diploma vs. 35% high-school-diploma vs. 49% university-degree; p < 0.01) and unstable employment (35% vs. 44%; p < 0.05). At multivariate logistic regression analysis, age <35 years (OR 1.61; 95%CI: 1.14-2.28) and lowest educational level (OR 1.64; 95% CI: 1.13-2.37, compared to University degree) were independently associated with non-adherence. CONCLUSION: Only 41% of women had postpartum T2DM screening. Women with lower attendance are those with age <35 years or low educational level. Further strategies are needed to implement postpartum test.

Early vs. standard screening and treatment of gestational diabetes in high-risk women - An attempt to determine relative advantages and disadvantages.

BACKGROUND AND AIMS: Screening for Gestational Diabetes (GDM) is usually recommended between 24 and 28 weeks of pregnancy; however available evidence suggests that GDM may be already present before recommended time for screening, in particular among high-risk women as those with prior GDM or obesity. The purpose of this retrospective study was to evaluate whether early screening (16-18 weeks) and treatment of GDM may improve maternal and fetal outcomes. METHODS AND RESULTS: In 290 women at high-risk for GDM, we analyzed maternal and fetal outcomes, according to early or standard screening and GDM diagnosis time. Early screening was performed by 50% of high-risk women. The prevalence of GDM was 62%. Among those who underwent early screened, GDM was diagnosed at the first evaluation in 42.7%. Women with early diagnosis were more frequently treated with insulin and had a slightly lower HbA1c than women with who were diagnosed late. No differences were observed in the prevalence of Cesarean section, operative delivery, gestational age at the delivery, macrosomia, neonatal weight, Ponderal Index and Large-for-Gestational-Age among women with early or late GDM diagnosis or NGT. However, compared to NGT women, GDM women, irrespective of the time of diagnosis, had a lower gestational weight gain, lower prevalence of macrosomia (3.9% vs. 11.4%), small (1.7% vs. 8.3%) as well as large for gestational age (3.3% vs. 16.7%), but higher prevalence of pre-term delivery (8.9% vs. 2.7%). CONCLUSION: Early vs. standard screening and treatment of GDM in high-risk women is associated with similar short-term maternal-fetal outcomes, although women with an early diagnosis were treated to a greater extent with insulin therapy.

The role of adipokines in the pathogenesis of gestational diabetes mellitus.

Gestational diabetes mellitus (GDM) is a complex condition whose physiopathology to date has not been completely clarified. Two major metabolic disorders, insulin resistance and ß-cells dysfunction, play currently major role in pathogenesis of GDM. These elements are influenced by the amount of adipose tissue present before and/or during the pregnancy. Consequently, adipokines (adiponectin (APN), leptin (LPT), adipocyte fatty acid-binding protein, resistin, visfatin, omentin, vaspin, apelin, chemerin) secreted by adipose tissue, may contribute directly and/or indirectly, through the enhancement of chronic inflammation, aggravating insulin resistance and promoting GDM onset. This review aims to outline the potential physiopathological and prognostic role in GDM of adipokines, mainly APN and LPT.

Review of general suggestions on physical activity to prevent and treat gestational and pre-existing diabetes during pregnancy and in postpartum.

The aim of this review is to provide general suggestions on physical activity (PA) in pre-gestational and gestational diabetes mellitus (GDM) and encourage women to take part in safe and effective activities throughout pregnancy, in the absence of other contraindications. PA before and during pregnancy and in postpartum has many positive effects on the mother, as it could reduce the risk of GDM, excessive weight gain and lower back pain and also prevents, in the postpartum, diabetes mellitus. It may also reduce the duration of labour and complications at childbirth, fatigue, stress, anxiety and depression, thereby leading to an improved sense of wellbeing. Clinically, it is thought to help prevent preeclampsia and premature birth even though RCTs provide conflicting evidence with regard to the prevention of GDM. The main reason for this rests on the fact that the majority of clinical trials have not been able to replicate the preventive effect of PA on the onset of GDM, such as the different adherence of the patient to PA. Herein, we survey the literature regarding exercise and PA on GDM prevention and treatment as well as on clinical outcomes in pre-GDM in pregnancy. On the basis of the current literature, we also present a series of general recommendations and suggestions on PA and exercise training in pregnancy among both diabetic patients and those at risk for GDM.

Study of the NR4A family gene expression in patients with multiple sclerosis treated with Fingolimod.

BACKGROUND AND PURPOSE: Fingolimod is a drug approved for treatment of relapsing-remitting multiple sclerosis (RRMS) that exerts its effects via sequestering lymphocytes within the lymph nodes. The drug, acting on the sphingosine-1-phosphate pathway, is involved in a plethora of processes and, to date, its mechanism of action is not completely understood. Recently, it has been demonstrated that Fingolimod increases the expression of transcription factor NR4A2 in murine brain. NR4A2 belongs to nuclear receptor family 4, group A (NR4A) along with NR4A1 and NR4A3. The role of NR4A2 in the pathogenesis of multiple sclerosis is already known and supported by its down-regulation observed in blood obtained from patients with RRMS compared with healthy controls (HCs). It is notable that NR4A2 impairment is reversed in patients with RRMS during pregnancy, which represents a transitory state of immune tolerance, associated with reduced disease activity. An inverse correlation between NR4A2 gene expression levels and clinical parameters indicates that more aggressive forms of the disease are characterized by lower levels of NR4A2. METHODS: Gene expression levels of NR4A in blood obtained from HCs, treatment-naive (T0) and Fingolimod-treated patients with RRMS were evaluated to determine their contribution to drug response. RESULTS: Gene expression levels of NR4A were down-regulated in T0 patients compared with HCs. Patients treated with Fingolimod for >2 years were characterized by higher levels of NR4A2 compared with the T0 group, approaching those of HCs. NR4A1 and NR4A3 levels were not altered. CONCLUSIONS: Involvement of the NR4A family in the pathogenesis of multiple sclerosis and a role of Fingolimod in the recovery from NR4A2 deficit can be hypothesized based on our data.

Two-year real-life efficacy, tolerability and safety of dimethyl fumarate in an Italian multicentre study.

BACKGROUND: Dimethyl-fumarate (DMF) demonstrated efficacy and safety in relapsing-remitting multiple sclerosis (MS) in randomized clinical trials. OBJECTIVES: To track and evaluate post-market DMF profile in real-world setting. MATERIALS AND METHODS: Patients receiving DMF referred to Italian MS centres were enrolled and prospectively followed, collecting demographic clinical and radiological data. RESULTS: Among the 735 included patients, 45.4% were naïve to disease-modifying therapies, 17.8% switched to DMF because of tolerance, 27.4% switched to DMF because of lack of efficacy, and 9.4% switched to DMF because of safety concerns. Median DMF exposure was 17 months (0-33). DMF reduced the annual relapse rate (ARR) by 63.2%. At 12 and 24 months, 85 and 76% of patients were relapse-free. NEDA-3 status after 12 months of DMF treatment was maintained by 47.5% of patients. 89 and 70% of patients at 12 and 24 months regularly continued DMF. Most frequent adverse events (AEs) were flushing (37.2%) and gastro-enteric AEs (31.1%). CONCLUSION: Our post-market study corroborated that DMF is a safe and effective drug. Additionally, the study suggested that naïve patients strongly benefit from DMF and that DMF improved ARR also in patients who were horizontally switched from injectable therapies due to tolerability and efficacy issues.

A multicenter, observational, prospective study of self- and parent-reported quality of life in adolescent multiple sclerosis patients self-administering interferon-ß1a using RebiSmart™-the FUTURE study.

Besides the impact of disease per se, the use of immunomodulatory therapies in adolescents with relapsing-remitting multiple sclerosis (RRMS) may have an effect on quality of life (QL). The FUTURE (Quality of liFe in adolescent sUbjecTs affected by mUltiple sclerosis treated with immunomodulatoRy agEnt using self-injecting device) study was designed to evaluate the changes in QL of Italian adolescents with RRMS receiving treatment with IFN-ß1a (Rebif; 22 µg), administered subcutaneously three times weekly using the RebiSmart™ electronic autoinjection device over a 52-week period. Fifty adolescents with RRMS were enrolled and 40 completed the study. Changes from baseline to end of treatment (EoT) in adolescent self-reported and parent-reported QL were assessed using the Pediatric Quality of Life Inventory Multidimensional Fatigue Scale (PedsQL), which has been validated for use in pediatric MS and for which an Italian version is available. The adolescent self-reported total PedsQL4.0 score and all of its subscales tended to increase from baseline to EoT, the only exception being "Emotional functioning." In parent-reported measures, the total PedsQL4.0 score increased significantly from baseline to EoT (+ 5.27 points, p = 0.041). Significant increases were also evident for parent-reported "Psychosocial health summary score" (+ 5.90 points; p = 0.015) and "School functioning" (+ 7.84 points; p = 0.029). Our results indicate that adolescents with RRMS using the electronic injection device RebiSmart™ for self-administration of Rebif® can experience long-term improvements in QL.

Italian national guidelines for the screening of gestational diabetes: Time for a critical appraisal?

BACKGROUND AND AIM: In 2011, the Italian National Health System guidelines introduced a selective screening for gestational diabetes (GDM) based on risk factors, recommending early evaluation in high risk women. The present study examined to which extent guidelines are applied, and analyzed the effectiveness of GDM diagnosis according to risk profile. METHODS AND RESULTS: We analyzed 1338 pregnant women, consecutively screened for GDM with a 75 g OGTT between January 2013 and December 2015, according to national guidelines. Diagnosis of GDM was based on IADPSG/WHO 2013 criteria. As many as 14.4% of screened women was at high risk, 64% at medium, 21.6% did not have any risk factor. Only 50% of high-risk women were appropriately screened at 16th-18th gestational weeks; 28% of them repeated the OGTT due to NGT. The overall prevalence of GDM was 39.9%, higher in high risk women (67% vs. 40% medium risk vs. 22% low risk; p < 0.0001). An early GDM diagnosis was performed in 40.7% of high-risk women. In low risk women, gestational weight gain at the screening time was independently associated with GDM. CONCLUSIONS: The recommendations for the screening of GDM are still insufficiently implemented, especially for early evaluation in high risk women. Considering the high proportion of early GDM diagnosis, the poor adherence to screening recommendation may result in late diagnosis of GDM. Finally, our finding of a 22% prevalence of GDM among low risk women suggests the need to consider additional risk factors, such as excessive weight gain during pregnancy.

Anti-inflammatory genes associated with multiple sclerosis: a gene expression study.

Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system caused by a complex interaction between multiple genes and environmental factors. HLA region is the strongest susceptibility locus, but recent huge genome-wide association studies identified new susceptibility genes. Among these, BACH2, PTGER4, RGS1 and ZFP36L1 were highlighted. Here, a gene expression analysis revealed that three of them, namely BACH2, PTGER4 and ZFP36L1, are down-regulated in MS patients' blood cells compared to healthy subjects. Interestingly, all these genes are involved in the immune system regulation with predominant anti-inflammatory role and their reduction could predispose to MS development.

Biological monitoring of IFN-ß therapy in Multiple Sclerosis.

Multiple Sclerosis (MS) is a heterogeneous disease and a variable percentage of patients are non-responders to common treatment. Early diagnosis of non-responders allows change to a more useful therapy for the patient and better allocates a large amount of financial resources. Quantification of Neutralizing antibodies (Nabs) and of biological activity of IFN-ß are recognized approaches to identify immuno-pharmacological non-responders. A consistent number of studies have demonstrated that quantification of Myxovirus-induced protein A (MxA) is a valid biomarker to detect immune-pharmacological non responders after one year of treatment. Persistent high titre of Nabs and absence of biological activity predict abolition of IFN-ß effects in disease activity measured through MRI, number of relapses and disability. Guidelines and flow-charts including both Nabs and MxA quantification are presented.

No impact of current therapeutic strategies on disease reactivation after natalizumab discontinuation: a comparative analysis of different approaches during the first year of natalizumab discontinuation.

BACKGROUND AND PURPOSE: Natalizumab discontinuation induces the recurrence of multiple sclerosis disease activity: currently no therapeutic approach has been found able to abolish disease reactivation. METHODS: The recurrence of disease activity after natalizumab discontinuation was retrospectively evaluated in 79 patients who had been treated with immunomodulating agents, other first-line therapies, fingolimod or not treated. RESULTS: No differences have been found in clinical or magnetic resonance imaging recurrence of disease activity amongst the groups. Interestingly, no disease reactivation was observed only in one patient treated for 6 months with monthly pulses of cyclophosphamide. CONCLUSION: Disease modifying treatment or 'no treatment' is unable to abolish disease activity reactivation after natalizumab discontinuation.

Multiple Sclerosis State of the Art (SMART): A Qualitative and Quantitative Analysis of Therapy's Adherence, Hospital Reliability's Perception, and Services Provided Quality.

The purpose of this study was to assess the adherence to therapy in patients with relapsing remitting multiple sclerosis (RR-MS) and to analyze the possible influence of factors such as hospital care and patients socioeconomic status. Two hundred eighty-five patients with RR-MS according to Mc Donald's criteria and naïve disease-modifying drugs (DMDs) naïve were enrolled. Two self-administered questionnaires addressing the management of patients at therapy prescription and the personal perception of the daily life changes caused by DMDs were administered at months 3 and 12. Full adherence, considered as correct use of the therapy prescribed, was observed in a very high percentage of subjects (97.3% and 93.9% at 3 and 12 months). The main cause for reduced adherence was single dose forgetfulness, followed by anxiety, pain at the injection site, and tiredness of "doing all injections." Nurses and neurologists of MS Center were identified as the major resource in coping with the disease at 3 and 12 months by patients. The neurologist was the health professional involved in MS management in 95% of cases and the nurse appeared to play a central role in patient training and drug administration management (50.3%).

Monocytes and CD4+ T cells contribution to the under-expression of NR4A2 and TNFAIP3 genes in patients with multiple sclerosis.

We recently found a gene signature for multiple sclerosis (MS) that reverted to normal during pregnancy in MS patients and included NR4A2 and TNFAIP3, key molecules in anti-inflammatory processes. Here we focus on the expression levels of these two genes in monocytes and CD4+ T cells from healthy controls and treatment-naïve RRMS patients. Our findings show that monocytes play a key role in the dysregulated anti-inflammatory response, being the expression of both genes down-regulated in these cells in RRMS patients with respect to healthy individuals. CD4+ T cells seem to have only a marginal part, because we can observe only a slight down-regulation in NR4A2.

Early detection of neutralizing antibodies to interferon-beta in multiple sclerosis patients: binding antibodies predict neutralizing antibody development.

BACKGROUND: Neutralizing antibodies (NAb) affect efficacy of interferon-beta (IFN-b) treatment in multiple sclerosis (MS) patients. NAbs evolve in up to 44% of treated patients, usually between 6-18 months on therapy. OBJECTIVES: To investigate whether early binding antibody (BAb) titers or different IFN-b biomarkers predict NAb evolution. METHODS: We included patients with MS or clinically isolated syndrome (CIS) receiving de novo IFN-b treatment in this prospective European multicenter study. Blood samples were collected at baseline, before and after the first IFN-b administration, and again after 3, 12 and 24 months on that therapy; for determination of NAbs, BAbs, gene expression of MxA and protein concentrations of MMP-9, TIMP-1, sTRAIL, CXCL-10 and CCL-2. RESULTS: We found that 22 of 164 (13.4%) patients developed NAbs during a median time of 23.8 months on IFN-b treatment. Of these patients, 78.9% were BAb-positive after 3 months. BAb titers ≥ 1:2400 predicted NAb evolution with a sensitivity of 74.7% and a specificity of 98.5%. Cross-sectionally, MxA levels were significantly diminished in the BAb/NAb-positive samples; similarly, CXCL-10 and sTRAIL concentrations in BAb/NAb-positive and BAb-positive/NAb-negative samples, respectively, were also diminished compared to BAb/NAb-negative samples. CONCLUSIONS: BAb titers reliably predict NAbs. CXCL-10 is a promising sensitive biomarker for IFN-b response and its abrogation by anti-IFN-b antibodies.

Observational case-control study of the prevalence of chronic cerebrospinal venous insufficiency in multiple sclerosis: results from the CoSMo study.

BACKGROUND: Chronic cerebrospinal venous insufficiency (CCSVI) has been proposed as a possible cause of multiple sclerosis (MS). OBJECTIVES: The CoSMo study evaluated the association between CCSVI and MS. METHODS: The primary end-point of this multicentric, case-control study was to compare the prevalence of CCSVI between patients with MS, patients with other neurodegenerative diseases (ONDs) and healthy controls (HCs). Color-coded duplex sonography was performed by a sonologist and the images were sent to one of three central sonologists for a second reading. Agreement between local and central sonologists or, in case of disagreement, the predominant judgment among the three central readers, was required for a diagnosis of CCSVI. All readings, data collection and analysis were blinded. RESULTS: The study involved 35 MS centers across Italy and included 1874 subjects aged 18-55. 1767 (94%) were evaluable: 1165 MS patients, 226 patients with ONDs and 376 HCs. CCSVI prevalence was 3.26%, 3.10% and 2.13% for the MS, OND and HC groups, respectively. No significant difference in CCSVI prevalence was found amongst the three cohorts (MS versus HC, OR = 1.55, 95%CI = 0.72-3.36, p = 0.30; OND versus HC, OR = 1.47, 95%CI = 0.53-4.11, p = 0.46; MS versus OND, OR = 1.05, 95%CI = 0.47-2.39, p = 0.99). High negative and low positive agreement was found between the local and centralized readers. CONCLUSIONS: CCSVI is not associated with MS.

Urinary JCV-DNA testing during natalizumab treatment may increase accuracy of PML risk stratification.

The risk of progressive multifocal leukoencephalopathy (PML) in patients treated with natalizumab for multiple sclerosis (MS) is a serious concern. The presence of anti-JC virus antibodies is a risk factor for PML development, but 2.5 % of the patients result falsely-negative, while the prognostic relevance of testing JCV-DNA in biological fluids of treated patients is debated. Aim of this work was to evaluate the utility of testing JCV-DNA, together with anti-JCV antibodies, in biological samples of treated patients as a tool for PML risk stratification. 126 subjects from 5 MS Centers in Italy were included in the study. We performed a cross-sectional study in 63 patients testing JCV-DNA in blood, peripheral blood cells and urine. We longitudinally assessed the presence of JCV-DNA in a cohort of 33 subjects, one of which developed PML. We could test retrospectively serum samples from another PML case occurred during natalizumab therapy. Anti-JCV antibodies and urinary JCV-DNA were both tested in 73 patients. No changes in JCV-DNA status occurred during natalizumab treatment. The subject who developed PML in the longitudinal cohort had detectable JCV-DNA in urine at all time-points while serum or blood from both PML patients were always negative before the onset of disease and, in one case, after. Four subjects with JCV-DNA in urine and undetectable anti-JCV antibodies were retested for anti-JCV antibodies and three out of four resulted positive. In conclusion, testing JCV-DNA in urine is complementary to testing anti-JCV antibodies in identifying patients at risk of PML.