Leukocyte Phosphomannomutase and Phosphomannose Isomerase Activity in an Indian Cohort.

Advances in molecular sequencing technology has increased the diagnostic yield for Congenital disorder of glycosylation (CDG). However, novel variants or those of uncertain significance (vus) often pose a challenge and in such cases confirmed diagnosis can be warranted through enzyme analysis of these defects. We thus, aimed to optimize leukocyte-based enzyme assays for first two enzymes involved in N-glycosylation pathway i.e. Phosphomannomutase (PMM) and Phosphomannose isomerase (MPI). Study population comprised of 50 healthy non-alcoholic adults and 20 pediatric controls. Leukocyte enzyme activity was measured by monitoring the conversion of NADP to NADPH at 340 nm. The conditions were optimized and precision was assessed for both low and normal activity leukocyte controls. Enzyme activities for PMM and MPI in healthy individuals were measured in the range 1.6-3.9 and 7-20 nmol/min/mg protein respectively and did not vary with age and gender. The precision for both PMM and MPI showed %CV of 19.9 and 19.8 respectively. The enzyme activity in leukocyte pellet was found to be stable for up to 9 months when stored at -80 °C. The enzyme assays are optimized for PMM and MPI and can be used for evaluation of CDG patients in India.

Optimization of infliximab therapy in inflammatory bowel disease using a dashboard approach-an Indian experience.

PURPOSE: Infliximab (IFX) therapy in inflammatory bowel disease (IBD) is associated with loss of response in half the patients, due to complex pharmacokinetic and immunological factors. Dashboard's Bayesian algorithms use information from model and individual multivariate determinants of IFX concentration and can predict dose and dosing interval. AIM: To compare measured IFX concentrations in our laboratory with values predicted by iDose dashboard system and report its efficacy in managing patients not responding to conventional dosing schedule. METHOD: Clinical history, demographic details, and laboratory findings such as albumin and C-reactive protein (CRP) data of IBD patients (n = 30; median age 23 years (IQR: 14.25 - 33.5)) referred for IFX drug monitoring in our laboratory from November 2017 to November 2019 were entered in iDose software. The IFX concentration predicted by iDose based on this information was compared with that measured in our laboratory. In addition, a prospective dashboard-guided dosing was prescribed in 11 of these 30 patients not responding to conventional dosing and was followed to assess their clinical outcome. RESULT: IFX monitoring in our 30 patients had shown therapeutic concentration in 12, supratherapeutic in 2 and subtherapeutic concentration in 16 patients. The iDose predicted concentration showed concordance in 21 of these 30 patients. Of 11 patients managed with iDose-assisted prospective dosing, 8 achieved clinical remission, 2 showed partial response, and one developed antibodies. CONCLUSION: Retrospective data analysis showed concordance between laboratory measured and iDose-predicted IFX level in 70% of patients. iDose-assisted management achieved clinical remission and cost reduction.

Clinical efficacy of infliximab level and anti-infliximab antibody measurement in patients with inflammatory bowel disease: An audit.

BACKGROUND AND AIMS: Infliximab (IFX) monitoring has been proposed for effective therapeutic management of inflammatory bowel disease (IBD). There is no data on infliximab levels and its antibody measurement in Indian patients. We assessed the clinical efficacy of IFX level and antibodies to infliximab (ATI) monitoring in IBD patients. METHODS: Infliximab trough level and antibody testing was done in 50 and 30 IBD patients, respectively using commercially available enzyme-linked immunosorbent assay (ELISA) kits. The levels were correlated with the disease status, albumin, and C-reactive protein (CRP) levels. The clinical efficacy of level-based change in patient management was evaluated. RESULTS: Of 50 patients, IFX levels were therapeutic in 8, sub-therapeutic in 40, and supra-therapeutic in 2. High ATI titer was present in 8/30 patients. The IFX level did not correlate with the dose of 5 or 10 mg/kg. Based on IFX level and ATI estimation, management was changed in 35 patients: increase in dose in 7, decrease in dosing interval in 17, increase in interval in 2, surgery in 2, change in biologic in 5, and cessation of IFX in 2 patients. Therapy modification based on IFX level improved the clinical response in 25 patients, of whom 5 are in remission at a median duration of 2 years. CONCLUSION: Most (80%) of the IBD patients had subtherapeutic IFX levels while high ATI titers were found in 27% of the patients. There was no correlation between infliximab dose and drug levels. Therapy modification based on drug level benefitted the majority. Our results suggest that measurement of IFX level assists in attaining therapeutic levels and improves clinical response.

Comparison of transferrin isoform analysis by capillary electrophoresis and HPLC for screening congenital disorders of glycosylation.

BACKGROUND: Transferrin, a major glycoprotein has different isoforms depending on the number of sialic acid residues present on its oligosaccharide chain. Genetic variants of transferrin as well as the primary (CDG) & secondary glycosylation defects lead to an altered transferrin pattern. Isoform analysis methods are based on charge/mass variations. We aimed to compare the performance of commercially available capillary electrophoresis CDT kit for diagnosing congenital disorders of glycosylation with our in-house optimized HPLC method for transferrin isoform analysis. METHODS: The isoform pattern of 30 healthy controls & 50 CDG-suspected patients was determined by CE using a Carbohydrate-Deficient Transferrin kit. The results were compared with in-house HPLC-based assay for transferrin isoforms. RESULTS: Transferrin isoform pattern for healthy individuals showed a predominant tetrasialo transferrin fraction followed by pentasialo, trisialo, and disialotransferrin. Two of 50 CDG-suspected patients showed the presence of asialylated isoforms. The results were comparable with isoform pattern obtained by HPLC. The commercial controls showed a <20% CV for each isoform. Bland Altman plot showed the difference plot to be within +1.96 with no systemic bias in the test results by HPLC & CE. CONCLUSION: The CE method is rapid, reproducible and comparable with HPLC and can be used for screening Glycosylation defects.