Comparison of live and fixed cell-based assay performance: implications for the diagnosis of MOGAD in a low-middle income country.

Background: Though considered optimal, live cell-based assay (LCBA) is often unavailable for the diagnosis of myelin oligodendrocyte glycoprotein antibody-associated disorders (MOGAD) in resource-poor regions. This study was undertaken to determine the agreement between LCBA and the widely available fixed cell-based assay (FCBA), for recommending testing guidelines within our region. Method: All consecutive patients in our registry with a MOGAD phenotype were tested. The results from a commercially available FCBA (Euroimmun, Germany) were compared with a validated "in-house" LCBA. Clinical and MRI data were available for correlation. Results: Among the 257 patient samples tested, 118 (45.9%) were positive by FCBA titre ≥1: 10 and or LCBA titres ≥1: 160 titre and 139 samples were negative. There was robust agreement between the two assays (agreement 98.8%, Cohen's kappa 0.98 [95% CI- 0.95-1.00], Spearman correlation 0.97 (p < 0.0001). Among five discordant samples, four had clinical and or MRI data which supported an alternate diagnosis. There was a modest correlation between assay titres, particularly for samples with titres ≥ 1:100 in FCBA (Spearman's Rho 0.26, p 0.005). Thirty samples were positive by FCBA at < 1:100 titre and included 1:80 (20),1:40(7) and 1:10 (3) titres. Among them, 80% had clear positive titres when tested by LCBA. Conclusion: The FCBA tested with serum dilutions of 1:10 was highly predictive of MOGAD in our study and compared well with our "in-house" LCBA. The current recommendations for testing at higher dilutions need to be re-examined in light of our findings. The results of our study should ideally be replicated in a larger dataset but at the same time provide some guidance for the accurate diagnosis of MOGAD in resource-poor settings.

Incidence and Prevalence of Neuromyelitis Optica Spectrum Disorders in the Background of International Consensus Diagnostic Criteria - A Systematic Review.

Introduction of international consensus criteria (2015 IPND criteria) for neuromyelitis optica spectrum disorders (NMOSDs) has improved diagnostic accuracy for aquaporin 4 (AQP4)-IgG-associated and seronegative NMOSDs. This study aimed to review relevant publications related to the incidence and prevalence of NMOSDs and provide an updated review of the global epidemiology of NMOSDs in the light of new diagnostic criteria. A comprehensive literature search was performed from January 2015 to June 2021 by using appropriate keywords in PubMed, Scopus, and Web of Science. Relevant papers that fulfilled inclusion criteria were shortlisted and reviewed. Twenty-one papers were selected for this review. Incidence of NMOSDs was 0.04-0.25/100,000 in predominantly white and 0.34-1.31/100,000 in nonwhite populations. Prevalence was 0.70-1.91/100,000 in white and 0.86-4.52/100,000 in nonwhite populations. The 2015 IPND criteria significantly improved the incidence and prevalence rates for NMOSDs when compared to the Wingerchuk 2006 criteria. Incidence of MOG-IgG-associated NMOSDs was 0.12-0.13/100,000, with prevalence in children 0.03-1.4/100,000 and in adults 0.65-2/100,000. In this systematic review, studies that used uniform diagnostic criteria and confirmed cases after testing for AQP4-IgG were included. The prevalence of NMOSDs was estimated to be <5/100,000 globally. A clear bias was seen in favor of nonwhite and indigenous populations. This review highlights the need for prospective population-based epidemiological studies and the importance of surveys in nonwhite populations around the globe.

Longitudinal Retinal Changes in MOGAD.

OBJECTIVE: Patients with myelin oligodendrocyte glycoprotein antibody (MOG-IgG)-associated disease (MOGAD) suffer from severe optic neuritis (ON) leading to retinal neuro-axonal loss, which can be quantified by optical coherence tomography (OCT). We assessed whether ON-independent retinal atrophy can be detected in MOGAD. METHODS: Eighty patients with MOGAD and 139 healthy controls (HCs) were included. OCT data was acquired with (1) Spectralis spectral domain OCT (MOGAD: N = 66 and HCs: N = 103) and (2) Cirrus high-definition OCT (MOGAD: N = 14 and HCs: N = 36). Macular combined ganglion cell and inner plexiform layer (GCIPL) and peripapillary retinal nerve fiber layer (pRNFL) were quantified. RESULTS: At baseline, GCIPL and pRNFL were lower in MOGAD eyes with a history of ON (MOGAD-ON) compared with MOGAD eyes without a history of ON (MOGAD-NON) and HCs (p < 0.001). MOGAD-NON eyes had lower GCIPL volume compared to HCs (p < 0.001) in the Spectralis, but not in the Cirrus cohort. Longitudinally (follow-up up to 3 years), MOGAD-ON with ON within the last 6-12 months before baseline exhibited greater pRNFL thinning than MOGAD-ON with an ON greater than 12 months ago (p < 0.001). The overall MOGAD cohort did not exhibit faster GCIPL thinning compared with the HC cohort. INTERPRETATION: Our study suggests the absence of attack-independent retinal damage in patients with MOGAD. Yet, ongoing neuroaxonal damage or edema resolution seems to occur for up to 12 months after ON, which is longer than what has been reported with other ON forms. These findings support that the pathomechanisms underlying optic nerve involvement and the evolution of OCT retinal changes after ON is distinct in patients with MOGAD. ANN NEUROL 2022;92:476-485.

Astrocytic outer retinal layer thinning is not a feature in AQP4-IgG seropositive neuromyelitis optica spectrum disorders.

BACKGROUND: Patients with anti-aquaporin-4 antibody seropositive (AQP4-IgG+) neuromyelitis optica spectrum disorders (NMOSDs) frequently suffer from optic neuritis (ON) leading to severe retinal neuroaxonal damage. Further, the relationship of this retinal damage to a primary astrocytopathy in NMOSD is uncertain. Primary astrocytopathy has been suggested to cause ON-independent retinal damage and contribute to changes particularly in the outer plexiform layer (OPL) and outer nuclear layer (ONL), as reported in some earlier studies. However, these were limited in their sample size and contradictory as to the localisation. This study assesses outer retinal layer changes using optical coherence tomography (OCT) in a multicentre cross-sectional cohort. METHOD: 197 patients who were AQP4-IgG+ and 32 myelin-oligodendrocyte-glycoprotein antibody seropositive (MOG-IgG+) patients were enrolled in this study along with 75 healthy controls. Participants underwent neurological examination and OCT with central postprocessing conducted at a single site. RESULTS: No significant thinning of OPL (25.02±2.03 µm) or ONL (61.63±7.04 µm) were observed in patients who were AQP4-IgG+ compared with patients who were MOG-IgG+ with comparable neuroaxonal damage (OPL: 25.10±2.00 µm; ONL: 64.71±7.87 µm) or healthy controls (OPL: 24.58±1.64 µm; ONL: 63.59±5.78 µm). Eyes of patients who were AQP4-IgG+ (19.84±5.09 µm, p=0.027) and MOG-IgG+ (19.82±4.78 µm, p=0.004) with a history of ON showed parafoveal OPL thinning compared with healthy controls (20.99±5.14 µm); this was not observed elsewhere. CONCLUSION: The results suggest that outer retinal layer loss is not a consistent component of retinal astrocytic damage in AQP4-IgG+ NMOSD. Longitudinal studies are necessary to determine if OPL and ONL are damaged in late disease due to retrograde trans-synaptic axonal degeneration and whether outer retinal dysfunction occurs despite any measurable structural correlates.

Retinal Optical Coherence Tomography in Neuromyelitis Optica.

BACKGROUND AND OBJECTIVES: To determine optic nerve and retinal damage in aquaporin-4 antibody (AQP4-IgG)-seropositive neuromyelitis optica spectrum disorders (NMOSD) in a large international cohort after previous studies have been limited by small and heterogeneous cohorts. METHODS: The cross-sectional Collaborative Retrospective Study on retinal optical coherence tomography (OCT) in neuromyelitis optica collected retrospective data from 22 centers. Of 653 screened participants, we included 283 AQP4-IgG-seropositive patients with NMOSD and 72 healthy controls (HCs). Participants underwent OCT with central reading including quality control and intraretinal segmentation. The primary outcome was thickness of combined ganglion cell and inner plexiform (GCIP) layer; secondary outcomes were thickness of peripapillary retinal nerve fiber layer (pRNFL) and visual acuity (VA). RESULTS: Eyes with ON (NMOSD-ON, N = 260) or without ON (NMOSD-NON, N = 241) were assessed compared with HCs (N = 136). In NMOSD-ON, GCIP layer (57.4 ± 12.2 µm) was reduced compared with HC (GCIP layer: 81.4 ± 5.7 µm, p < 0.001). GCIP layer loss (-22.7 µm) after the first ON was higher than after the next (-3.5 µm) and subsequent episodes. pRNFL observations were similar. NMOSD-NON exhibited reduced GCIP layer but not pRNFL compared with HC. VA was greatly reduced in NMOSD-ON compared with HC eyes, but did not differ between NMOSD-NON and HC. DISCUSSION: Our results emphasize that attack prevention is key to avoid severe neuroaxonal damage and vision loss caused by ON in NMOSD. Therapies ameliorating attack-related damage, especially during a first attack, are an unmet clinical need. Mild signs of neuroaxonal changes without apparent vision loss in ON-unaffected eyes might be solely due to contralateral ON attacks and do not suggest clinically relevant progression but need further investigation.

Overcoming the challenges in diagnosis of AQP4-IgG positive neuromyelitis optica spectrum disorders in resource poor settings using an indigenized and cost effective cell based assay.

BACKGROUND: Diagnosis of neuromyelitis optica spectrum disorders (NMOSD) in India is hindered by limited access to cost effective and sensitive assays for detection of aquaporin-4 antibody (AQP4-IgG) in India. OBJECTIVE: To develop a cost effective, sensitive, cell based assay (CBA) for detection of AQP4-IgG and to evaluate the serological status in patients with NMOSD diagnosed by 2015 diagnostic criteria. METHOD: Stably transfected Chinese hamster ovary (CHO) cell line expressing aquaporin M23 isomer was established. A fixed CBA was developed and validated in 381 samples including clinically definite NMOSD (n = 87), high risk NMOSD (n = 51), other demyelinating disorders (n = 92), other neurological disorders (n = 51) and healthy volunteers (n = 100). We tested the same samples again using a commercially available CBA and compared the results. All assays were performed by 2 independent investigators blinded to clinical and serological status. RESULTS: Our "in house"(Mangalore) assay showed sensitivity of 81.6% (95% CI 71.86-89.11%) for clinically definite NMOSD and 29.41% (95% CI 17.50-43.8%) for high risk NMOSD. Specificity was 100% for both groups. Both assays showed similar results for 67/ 87 (77.01%) patients with definite NMOSD while 4 samples tested positive by our assay alone (Cohen's kappa coefficient [K] - 0.86). Among the high risk group 14/51 (27.5%) samples showed similar results, one patient additionally was positive by the Mangalore assay (K - 0.95).

Role of Viral Infections in Multiple Sclerosis Pathogenesis among Indian Population.

BACKGROUND: The role of viral infections in multiple sclerosis (MS) pathogenesis is unclear. OBJECTIVE: Certain neurotropic viruses previously linked with MS among white population were studied including Epstein-Barr virus, human herpesvirus-6 (HHV-6) and MS-associated retrovirus (MSRV). MATERIAL AND METHODS: Sixty-two MS patients (37 had a recent clinical relapse) and 65 controls with other neurological disorders were included. Blood and cerebrospinal fluid (CSF) samples were obtained and processed with the primary objective of determining whether there was intrathecal multiplication of viruses under study (EBV, HHV6 A and B and human endogenous retrovirus) or a breach in blood-brain barrier associated with viral presence in both peripheral blood and CSF. RESULTS: Evidence of breach in blood-brain barrier was seen in 86.5% of patients as evidenced by abnormal CSF/serum albumin index and or MRI. EBV nuclear antigen (EBNA1 IgG) was seen in 89% of MS patients and 58% controls (P = <0.001). However, HHV6 IgG was similar in both groups (85% versus 81%; P = 0.45). In affinity immunoblotting reaction intrathecal IgG synthesis against EBNA1 antigen was demonstrable in 26% (16/62) of patients and none against HHV6. A subset of patients showed significant elevation in mean copy number of plasma EBV DNA during relapse and there was a trend for the same among patients harboring HHV-6B. No evidence of isolated intrathecal viral presence or multiplication was seen. CONCLUSIONS: The results of our study suggest that viruses studied namely EBV and HHV6 have a role in triggering relapses through a peripheral mechanism, rather than a direct role through intrathecal multiplication.

Clostridium bolteae is elevated in neuromyelitis optica spectrum disorder in India and shares sequence similarity with AQP4.

OBJECTIVE: To understand the role of gut microbiome in influencing the pathogenesis of neuromyelitis optica spectrum disorders (NMOSDs) among patients of south Indian origin. METHODS: In this case-control study, stool and blood samples were collected from 39 patients with NMOSD, including 17 with aquaporin 4 IgG antibodies (AQP4+) and 36 matched controls. 16S ribosomal RNA (rRNA) sequencing was used to investigate the gut microbiome. Peripheral CD4+ T cells were sorted in 12 healthy controls, and in 12 patients with AQP4+ NMOSD, RNA was extracted and immune gene expression was analyzed using the NanoString nCounter human immunology kit code set. RESULTS: Microbiota community structure (beta diversity) differed between patients with AQP4+ NMOSD and healthy controls (p < 0.001, pairwise PERMANOVA test). Linear discriminatory analysis effect size identified several members of the microbiota that were altered in patients with NMOSD, including an increase in Clostridium bolteae (effect size 4.23, p 0.00007). C bolteae was significantly more prevalent (p = 0.02) among patients with AQP4-IgG+ NMOSD (n = 8/17 subjects) compared with seronegative patients (n = 3/22) and was absent among healthy stool samples. C bolteae has a highly conserved glycerol uptake facilitator and related aquaporin protein (p59-71) that shares sequence homology with AQP4 peptide (p92-104), positioned within an immunodominant (AQP4 specific) T-cell epitope (p91-110). Presence of C bolteae correlated with expression of inflammatory genes associated with both innate and adaptive immunities and particularly involved in plasma cell differentiation, B cell chemotaxis, and Th17 activation. CONCLUSION: Our study described elevated levels of C bolteae associated with AQP4+ NMOSD among Indian patients. It is possible that this organism may be causally related to the immunopathogenesis of this disease in susceptible individuals.

Genetic variations in the Dravidian population of South West coast of India: Implications in designing case-control studies.

BACKGROUND & OBJECTIVES: Indian data have been largely missing from genome-wide databases that provide information on genetic variations in different populations. This hinders association studies for complex disorders in India. This study was aimed to determine whether the complex genetic structure and endogamy among Indians could potentially influence the design of case-control studies for autoimmune disorders in the south Indian population. METHODS: A total of 12 single nucleotide variations (SNVs) related to genes associated with autoimmune disorders were genotyped in 370 healthy individuals belonging to six different caste groups in southern India. Allele frequencies were estimated; genetic divergence and phylogenetic relationship within the various caste groups and other HapMap populations were ascertained. RESULTS: Allele frequencies for all genotyped SNVs did not vary significantly among the different groups studied. Wright's FSTwas 0.001 per cent among study population and 0.38 per cent when compared with Gujarati in Houston (GIH) population on HapMap data. The analysis of molecular variance results showed a 97 per cent variation attributable to differences within the study population and <1 per cent variation due to differences between castes. Phylogenetic analysis showed a separation of Dravidian population from other HapMap populations and particularly from GIH population. INTERPRETATION & CONCLUSIONS: Despite the complex genetic origins of the Indian population, our study indicated a low level of genetic differentiation among Dravidian language-speaking people of south India. Case-control studies of association among Dravidians of south India may not require stratification based on language and caste.

Serological markers associated with neuromyelitis optica spectrum disorders in South India.

BACKGROUND: Neuromyelitis optica spectrum disorders (NMOSDs) represent 20% of all demyelinating disorders in South India. No studies have determined the seroprevalence to both antibodies against aquaporin-4* and antimyelin oligodendrocyte glycoprotein antibody (anti-MOG+) in this population. OBJECTIVE: To identify and characterize seropositive patients for anti-aquaporin-4 antibody (anti-AQP4+) and anti-MOG+ in South India. MATERIALS AND METHODS: We included 125 consecutive patients (15 children) who were serologically characterized using live transfected cells to human M23-AQP4 or full-length MOG. RESULTS: Among a total of 125 patients, 30.4% of patients were anti-AQP4+, 20% were anti-MOG+, and 49.6% were seronegative. No patient was positive for both. Anti-MOG+ patients represented 28.7% (25/87) of seronegative NMOSD. In comparison to anti-AQP4+ patients, anti-MOG+ patients were commonly male, had less frequent attacks and milder disability on expanded disability status score scale. Seronegative patients were also predominantly male, 36% (9/25) had monophasic longitudinally extensive transverse myelitis and disability was comparable with anti-AQP4+ patients. Lumbar cord involvement was common in anti-MOG+ and seronegatives, whereas anti-AQP4+ patients had more cervical lesions. CONCLUSION: Anti-AQP4+/anti-MOG + patients accounted for nearly half of the patients suspected of having NMOSD in South India, indicating that antibody testing may be useful on the management of subgroups with different prognosis.

European multiple sclerosis risk variants in the south Asian population.

BACKGROUND: In less than a decade, genomewide association studies have identified over 100 single-nucleotide variants that are associated with increased risk of developing multiple sclerosis. However, since these studies have focused almost exclusively on European populations, it is unclear what role these variants might play in determining risk in other ethnic groups. OBJECTIVE: To assess the effects of European multiple sclerosis-associated risk variants in the south Asian population. METHODS: Using a combination of chip-based genotyping and next-generation sequencing, we have assessed 109 European-associated variants in a total of 270 cases and 555 controls from the south Asian population. RESULTS: We found that two-thirds of the tested variants (72/109) showed over representation of the European risk allele in south Asian cases (p < 0.0003). In the rest of the Immunochip array, the most associated variant was rs7318477 which maps close to TNFSF13B, the gene for the B-cell-related protein BAFF. CONCLUSION: Our data indicate substantial overlap in genetic risk architecture between Europeans and south Asians and suggest that the aetiology of the disease may be largely independent of ethnicity.

Relapsing optic neuritis and isolated transverse myelitis are the predominant clinical phenotypes for patients with antibodies to myelin oligodendrocyte glycoprotein in India.

BACKGROUND: Clinical phenotypes of patients with antibodies to myelin oligodendrocyte glycoprotein (anti-MOG+) are unknown in India. OBJECTIVES: Retrospectively to characterise anti-MOG+ patients with inflammatory central nervous system disorders in India. METHOD: A total of 87 patients with non-multiple sclerosis demyelinating disorders (excluding acute disseminated encephalomyelitis) who were seronegative for anti-aquaporin 4 antibody were retrospectively analysed using a cell-based assay for anti-MOG+ status. RESULTS: Twenty-five patients were anti-MOG+ in this cohort. They represented 28.7% (25/87) of patients who tested negative for anti-AQP4+. Sixty-four per cent (16/25) of anti-MOG+ patients were men and had a relapsing course. Patients with recurrent optic neuritis and those with a single attack of acute longitudinally extensive transverse myelitis were the most common phenotypes. CONCLUSION: Relapsing optic neuritis was the most common phenotype, contrasting with a lower risk of relapses in transverse myelitis.

HLA associations in South Asian multiple sclerosis.

BACKGROUND: Previous efforts to identify Human Leukocyte Antigen (HLA) gene associations with multiple sclerosis (MS) in the South Asian population have been underpowered. AIM: To identify the primary HLA class II alleles associated with MS in Indians. METHODS: We typed HLA-DRB1, -DQA1 and -DQB1 in 419 patients and 451 unrelated controls by polymerase chain reaction using sequence specific oligonucleotide probes (PCR-SSOP). RESULTS: At the gene level DRB1 showed significant evidence of association (p=0.0000012), DQA1 showed only marginal evidence of association (p=0.04) and there was no evidence for association at DQB1 (p=0.26). At the DRB1 locus association is confirmed with the *15:01 (p=0.00002) and the *03 (p=0.00005) alleles. CONCLUSION: Our study confirms that the risk effects attributable to the HLA- DRB1*15:01and DRB1*03 alleles seen in Europeans are also seen in Indians. The absence of any evidence of association with DQB1 alleles reflects the lower linkage disequilibrium between DQB1 alleles and DRB1 risk alleles present in this population, and illustrates the potential value of fine mapping signals of association in different ethnic groups.

Mycophenolate mofetil in the treatment of multiple sclerosis: a preliminary report.

BACKGROUND: Mycophenolate mofetil (MMF) is an affordable and tolerable drug reported to be beneficial in the treatment of multiple sclerosis (MS). AIM: To determine efficacy of MMF as first line disease modifying drug (DMD) in 40 patients with MS seen in our demyelinating disease registry. MATERIALS AND METHODS: The annualized relapse rate (ARR) for 1 year prior to starting MMF therapy and 1 year post treatment was calculated. Pre- and post-treatment expanded disability status scores (EDSS), age at onset of treatment, disease duration, and type of MS were recorded. Wilcoxon rank sum test was used for comparison of ARRs and EDSS before and after treatment. RESULTS: Forty patients included 27 females and 13 males. Mean duration of MMF therapy was 24 months (range 14-33 months). Pre-treatment mean ARR of 0.95 was significantly different from post treatment mean ARR of 0.11 (P=0.0001). Pre-treatment mean EDSS 3.80 (inter quartile range [IQR] 3.5-4.5) was significantly different from post-treatment mean EDSS 2.66 (IQR 1.5-3.0, P=0.0001). No adverse effects were reported that required stopping of medication. Five patients discontinued treatment 6-11 months after starting therapy, two of whom relapsed subsequently. CONCLUSION: Our preliminary results support the use of MMF, a cheap and well-tolerated drug, as first line disease modifying drug in MS. Long-term results in a larger patient cohort is required for validating our preliminary conclusions.

Association of vitamin D and multiple sclerosis in India.

BACKGROUND: Vitamin D deficiency is widely prevalent in India. The association between vitamin D status and multiple sclerosis (MS) has not been previously studied in Indians. OBJECTIVE: The objective of this paper is to determine whether vitamin D status is associated with MS in India. METHODS: In this study 110 MS patients and 108 matched controls were included. Serum 25-hydroxyvitamin D (25(OH)D) was measured in 63 patients in relapse, 77 patients in remission and all controls. Quantity of sun exposure in childhood and body mass index (BMI) were calculated. Patients and controls were genotyped for HLA-DRB1*1501. RESULTS: Patients had significantly lower 25(OH)D levels than matched controls (p = 0.003), and patients in relapse had a significantly lower vitamin D level as compared to those in remission (p = 0.001). Vitamin D deficiency (< 50 nmol/l) was seen in a higher proportion of cases (71.8%) than controls (53.7%) (p = 0.01). Higher quartiles of vitamin D (> 58 nmol/l) showed an inverse relationship with MS (OR = 0.28, CI = 0.11-0.68, p= 0.005). This effect persisted after adjusting for sun exposure. CONCLUSION: The results of our study indicated that serum 25(OH)D shows an inverse relationship with MS in the Indian population. Reverse causality cannot be excluded.