An immunoassay that distinguishes real neuromyelitis optica signals from a labeling detected in patients receiving natalizumab

BACKGROUND: Cell-based assays for neuromyelitis optica (NMO) diagnosis are the most sensitive and specific methods to detect anti-aquaporin 4 (AQP4) antibodies in serum, but some improvements in their quantitative and specificity capacities would be desirable. Thus the aim of the present work was to develop a sensitive quantitative method for detection of anti-AQP4 antibodies that allows clear diagnosis of NMO and distinction of false labeling produced by natalizumab treatment. METHODS: Sera from 167 individuals, patients diagnosed with NMO (16), multiple sclerosis (85), optic neuritis (24), idiopathic myelitis (21), or other neurological disorders (13) and healthy controls (8), were used as the primary antibody in an immunofluorescence assay on HEK cells transfected with the M23 isoform of human AQP4 fused with enhanced green fluorescent protein. Cells used were freshly transfected or stored frozen and then thawed just before adding the serum. RESULTS: Microscopic observation and fluorescence quantification produced similar results in fresh and frozen samples. Serum samples from patients diagnosed with NMO were 100% positive for anti-AQP4 antibodies, while all the other sera were negative. Using serum from patients treated with natalizumab, a small and unspecific fluorescent signal was produced from all HEK cells, regardless of AQP4 expression. CONCLUSIONS: Our cell-based double-label fluorescence immunoassay protocol significantly increases the signal specificity and reduces false diagnosis of NMO patients, especially in those receiving natalizumab treatment. Frozen pretreated cells allow faster detection of anti-AQP4 antibodies

Comparative Analysis for the Presence of IgG Anti-Aquaporin-1 in Patients with NMO-Spectrum Disorders

Detection of IgG anti-Aquaporin-4 (AQP4) in serum of patients with Neuromyelitis optica syndrome disorders (NMOSD) has improved diagnosis of these processes and differentiation from Multiple sclerosis (MS). Recent findings also claim that a subgroup of patients with NMOSD, serum negative for IgG-anti-AQP4, present antibodies anti-AQP1 instead. Explore the presence of IgG-anti-AQP1 using a previously developed cell-based assay (CBA) highly sensitive to IgG-anti-AQP4. Serum of 205 patients diagnosed as NMOSD (8), multiple sclerosis (94), optic neuritis (39), idiopathic myelitis (29), other idiopathic demyelinating disorders of the central nervous system (9), other neurological diseases (18) and healthy controls (8), were used in a CBA over fixed HEK cells transfected with hAQP1-EGFP or hM23-AQP4-EGFP, treated with Triton X-100 and untreated. ELISA was also performed. Analysis of serum with our CBA indicated absence of anti-AQP1 antibodies, whereas in cells pretreated with detergent, noisy signal made reliable detection impossible. ELISA showed positive results in few serums. The low number of NMOSD serums included in our study reduces its power to conclude the specificity of AQP1 antibodies as new biomarkers of NMOSD. Our study does not sustain detection of anti-AQP1 in serum of NMOSD patients but further experiments are expected.Grants from “La Junta de Andalucía, Consejería de Innovación Ciencia y Empresa” (P08-CTS-03574) and Consejería de Salud (PI0298-2010), and from the “Instituto de Salud Carlos III” (Exp. PI12/01882) to Miriam Echevarría funded this work. We thank Genzyme Foundation in multiple sclerosis for giving to Miriam Echevarría one of their 2012 fellowships. We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI

Comparative analysis for the presence of IgG anti-aquaporin-1 in patients with NMO-Spectrum disorders

Detection of IgG anti-Aquaporin-4 (AQP4) in serum of patients with Neuromyelitis optica syndrome disorders (NMOSD) has improved diagnosis of these processes and differentiation from Multiple sclerosis (MS). Recent findings also claim that a subgroup of patients with NMOSD, serum negative for IgG-anti-AQP4, present antibodies anti-AQP1 instead. Explore the presence of IgG-anti-AQP1 using a previously developed cell-based assay (CBA) highly sensitive to IgG-anti-AQP4. Serum of 205 patients diagnosed as NMOSD (8), multiple sclerosis (94), optic neuritis (39), idiopathic myelitis (29), other idiopathic demyelinating disorders of the central nervous system (9), other neurological diseases (18) and healthy controls (8), were used in a CBA over fixed HEK cells transfected with hAQP1-EGFP or hM23-AQP4-EGFP, treated with Triton X-100 and untreated. ELISA was also performed. Analysis of serum with our CBA indicated absence of anti-AQP1 antibodies, whereas in cells pretreated with detergent, noisy signal made reliable detection impossible. ELISA showed positive results in few serums. The low number of NMOSD serums included in our study reduces its power to conclude the specificity of AQP1 antibodies as new biomarkers of NMOSD. Our study does not sustain detection of anti-AQP1 in serum of NMOSD patients but further experiments are expected

Predictive Value of Serum Antibodies and Point Mutations of AQP4, AQP1 and MOG in A Cohort of Spanish Patients with Neuromyelitis Optica Spectrum Disorders

The detection of IgG aquaporin-4 antibodies in the serum of patients with Neuromyelitis optica (NMO) has dramatically improved the diagnosis of this disease and its distinction from multiple sclerosis. Recently, a group of patients have been described who have an NMO spectrum disorder (NMOsd) and who are seronegative for AQP4 antibodies but positive for IgG aquaporin-1 (AQP1) or myelin oligodendrocyte glycoprotein (MOG) antibodies. The purpose of this study was to determine whether AQP1 and MOG could be considered new biomarkers of this disease; and if point mutations in the gDNA of AQP4, AQP1 and MOG genes could be associated with the etiology of NMOsd. We evaluated the diagnostic capability of ELISA and cell-based assays (CBA), and analyzed their reliability, specificity, and sensitivity in detecting antibodies against these three proteins. The results showed that both assays can recognize these antigen proteins under appropriate conditions, but only anti-AQP4 antibodies, and not AQP1 or MOG, appears to be a clear biomarker for NMOsd. CBA is the best method for detecting these antibodies; and serum levels of AQP4 antibodies do not correlate with the progression of this disease. So far, the sequencing analysis has not revealed a genetic basis for the etiology of NMOsd, but a more extensive analysis is required before definitive conclusions can be drawn.Ministerio de Economía y CompetitividadFEDER (Grants PI16/01249 y PI16/00493

Lack of impact of protease inhibitor resistance-associated mutations on the outcome of HIV-1-infected patients switching to darunavir-based dual therapy

[Background]: Little is known about the impact of baseline resistance-associated mutations (RAMs) on the outcomes of alternative therapeutic strategies such as dual regimens. We assessed the efficacy of boosted darunavir plus raltegravir (DRV + RAL) dual regimen as a simplification strategy in virologically suppressed patients with protease inhibitors RAMs.[Methods]: Retrospective, multicentre study on the evolution of 228 heavily pretreated patients who switched to boosted DRV + RAL according to genotypic sensitivity score (GSS). Patients were classified as full susceptible (GSS = 2; n = 177), or with reduced darunavir susceptibility (GSS < 2; n = 51).[Results]: Median (range) number of prior antiretroviral regimens was 9 (6–14), with a median (range) of 2 (1–3), 4 (3–6), and 5 (2–9) major mutations to non-nucleoside reverse transcriptase inhibitors, nucleoside reverse transcriptase inhibitors, and protease inhibitors, respectively. The median time of virological suppression before simplification was 49 months (IQR 39.8–63.5). Patients with reduced darunavir GSS showed a higher number of protease inhibitors-RAMs (9.3 vs 4.5, p < .01) and were suppressed for longer time (median, 61 months). At week 96, the rate of virological failure was low (two cases, 0.9%; 95% confidence interval, CI, 0.4–2.7%), and the efficacy, excluding non-virological reasons, was 96.8% (95%CI, 90.2–98.4%), without differences according to GSS or protease inhibitors-RAMs. Furthermore, significant improvements in CD4+ counts and CD4/CD8 ratio were observed (p < .01) in both groups.[Conclusions]: Treatment simplification to a dual regimen of boosted DRV + RAL after long-term virological suppression was not associated with a high risk of treatment failure, even in patients harbouring protease inhibitors-resistant HIV infection.This study was supported by the MSD Investigator-Initiated Studies Program (MISP) under grant number 57180

Maintenance of virologic suppression and improvement in comorbidities after simplification to raltegravir plus boosted darunavir among treatment-experienced HIV-infected patients

BIRDi study group.The use of two potent, well-tolerated, drugs could permit the maintenance of virologic suppression even in heavily pretreated people living with HIV. In this retrospective, multicenter, simplification study (NCT03348449), we included those patients with virologic suppression who switched to raltegravir (RAL) plus boosted darunavir (b/DRV). Overall, 345 patients (75 females, 25%) were included. Patients were largely pretreated (mean, 9.4 regimens), suppressed for a median of 41.1 months. Fifty patients had ≥1 mutation against DRV. At 96 weeks, the efficacy by intention-to-treat analysis (snapshot) was 73% (95%CI, 68.4–77.8%), but 97.1% (95%CI, 95.4–98.9) excluding changes due to non-virologic reasons, and virologic failure was rare (0.9%; 95%CI, 0.1–1.2%). Median CD4/CD8 ratio increased from 0.59 to 0.62 (p < 0.01), CD4+ cell count by +90 cells/µl (p < 0.01), and mean estimated glomerular filtration rate (eGFR) increased from 85.2 to 88.5 ml/min at 96 weeks, greater for patients receiving tenofovir disoproxil fumarate (eGFR, +3.6 ml/min, p = 0.04; serum phosphate +0.33 mg/dl; p < 0.01). There was a continued and significant improvement in the total cholesterol/high-density lipoprotein-cholesterol ratio. In conclusion, the simplification to a dual regimen with the combination of RAL and b/DRV is associated with maintenance of virologic suppression, even in largely pretreated patients, with improvements in CD4+ cell count, CD4/CD8 ratio, and in renal and lipid parameters