Clinical value of cell-based assays in the characterisation of seronegative myasthenia gravis.

OBJECTIVE: Patients with myasthenia gravis without acetylcholine receptor (AChR) or muscle-specific kinase (MuSK) antibodies detected by radioimmunoprecipitation assays (RIAs) are classified as seronegative myasthenia gravis (SNMG). Live cell-based assays (l-CBAs) can detect additional antibodies to clustered AChR, MuSK and low-density lipoprotein receptor-related protein 4 (LRP4), but positivity rates are variable and both clinical relevance and utility of CBA platforms remain unclear. METHODS: Sera from 82 patients with SNMG were tested by l-CBAs. Human embryonic kidney cells were transfected to individually express clustered AChR, MuSK or LRP4; or transfected to jointly express both clustered adult AChR and MuSK. Sera from 30 and 20 patients positive by RIA for AChR or MuSK antibodies were used as comparators. RESULTS: 53 of 82 (72%) patients with SNMG had generalised and 29 (28%) had ocular disease. The clustered AChR CBA detected antibodies in 16 of 82 patients (19.5%; including 4 patients with solely fetal AChR antibodies), while 7 of 82 (8.5%) patients had MuSK antibodies. A novel exploratory combined adult AChR-MuSK l-CBA efficiently detected all these antibodies in a subset of the SNMG cohort. No LRP4 antibodies were identified. Overall, patients with SNMG with clustered AChR antibodies, CBA-positive MuSK-MG or triple seronegative were younger, had less severe disease than patients with RIA-positive MG and had a better clinical outcome when immunotherapy was started soon after disease onset, although the time interval from onset to immunotherapy was not different when compared with patients with RIA-positive MG. CONCLUSION: Around one-third of patients with SNMG had AChR or MuSK antibodies by l-CBAs, which were efficiently detected with a combined l-CBA. The results in this large and unselected cohort of patients with MG demonstrate the diagnostic usefulness of performing CBAs and the importance of making these tests more widely available.

Safety and tolerability of SARS-Cov-2 vaccination in patients with myasthenia gravis: A multicenter experience.

BACKGROUND AND PURPOSE: During the COVID-19 pandemic, myasthenia gravis (MG) patients have been identified as subjects at high risk of developing severe COVID-19, and thus were offered vaccination with priority. The lack of direct data on the safety and tolerability of SARS-CoV-2 vaccines in MG have contributed to vaccine hesitancy. To address this issue, the safety and tolerability of SARS-CoV-2 vaccines were assessed in a large cohort of MG patients from two referral centers. METHODS: Patients with confirmed MG diagnosis, consecutively seen between October and December 2021 at two MG centers, were enrolled. Demographics, clinical characteristics, and information regarding SARS-CoV-2 infection/vaccination were extracted from medical reports and/or collected throughout telephonic or in-person interviews. RESULTS: Ninety-eight (94.2%) of 104 patients included were administered at least two vaccine doses 4 weeks before the interview or earlier, and among them, 63 of 98 (64.2%) have already received the "booster" dose. The most frequently used vaccines were BNT162b2-Pfizer-BioNTech and mRNA-1273-Moderna. Overall, only minor side effects were reported, most commonly local pain and fever. MG worsening after vaccination was observed in eight of 104 (7.7%) cases. The frequency of worsening among muscle-specific tyrosine kinase MG cases (3/9, 33.3%) was significantly higher compared to other serological subgroups. Spontaneous symptom regression was observed in six of eight cases. Twelve of 104 (11.5%) patients had SARS-CoV-2 infection, and none of the SARS-CoV-2-infected MG patients worsened after vaccination. CONCLUSIONS: Our data support the safety and tolerability of mRNA COVID-19 vaccines, which should be strongly recommended in MG patients, who could be at higher risk of complications if exposed to SARS-CoV-2 infection.

Cancer detection after a 9-year course of Lambert-Eaton myasthenic syndrome complicated by anti-Hu associated limbic encephalitis.

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune neuromuscular junction disorder, paraneoplastic in 55% of cases and commonly associated with small-cell lung cancer (SCLC). We report the case of a 61-year-old man presented who with a 3-month history of lower limb proximal weakness, progressing to upper limbs, associated with dysphagia, xerostomia and erectile dysfunction. Electrodiagnostic studies and anti voltage-gated calcium channel (VGCC) antibodies (Abs) detection confirmed LEMS diagnosis. Contrast-enhanced thorax computed tomography (CT) scan and subsequently [18F]-fluorodeoxyglucose (FDG) positron emission tomography (PET) showed no malignancy. Two years after the onset of LEMS, he was diagnosed with anti-Hu limbic encephalitis (LE). FDG-PET/CT scan remained negative for the following seven years. Nine years after LEMS onset, a hypermetabolic lesion of the left lung hilus was detected. This is a case of a paraneoplastic LEMS where the interval between the onset of neurological disease and tumour detection was as long as nine years.

Clinical, neurophysiological and serological clues for the diagnosis of neuromyotonia and distinction from cramp-fasciculation syndrome.

Neuromyotonia and cramp-fasciculation syndrome diagnosis currently relies on neurophysiological examination. In this study we investigated the clinical features and neural antibody profile of patients with neuromyotonia and cramp-fasciculation syndrome to assess the diagnostic value of serological testing. Available sera from adult patients with electromyography-defined neuromyotonia and cramp-fasciculation syndrome were tested for neural antibodies by indirect immunofluorescence on mouse brain sections and live cell-based assays. Forty patients were included, 14 with neuromyotonia and 26 with cramp-fasciculation syndrome. Neural antibodies were detected in 10/10 neuromyotonia sera, most commonly against contactin-associated protein 2 (7/10, 70%), and in 1/20 (5%) cramp-fasciculation syndrome sera. Clinical myokymia, hyperhidrosis, and paresthesia or neuropathic pain were more common in neuromyotonia and mostly associated with contactin-associated protein 2 antibodies. Central nervous system involvement was present in 4/14 (29%) neuromyotonia patients. A tumor was detected in 13/14 (93%) neuromyotonia patients (thymoma, 13), and in 4/26 (15%) with cramp-fasciculation syndrome (thymoma, 1; other neoplasms, 3). Twenty-one/27 (78%) patients achieved a significant improvement or complete remission. Our findings highlight clinical, neurophysiological and serological clues that can be useful in the diagnosis of neuromyotonia and cramp-fasciculation syndrome. Antibody testing is valuable for neuromyotonia diagnosis, while its usefulness in cramp-fasciculation syndrome confirmation is limited.

Comparison of Fixed and Live Cell-Based Assay for the Detection of AChR and MuSK Antibodies in Myasthenia Gravis.

BACKGROUND AND OBJECTIVES: Live cell-based assay (CBA) can detect acetylcholine receptors (AChRs) or muscle-specific tyrosine kinase (MuSK) antibodies (Abs) in a proportion of patients with radioimmunoassay (RIA)-double seronegative myasthenia gravis (dSN-MG). A commercial fixed CBA for AChR and MuSK Abs has recently become available; however, comparative studies on fixed and live CBAs are lacking. In this study, we compared the performance of fixed and live CBAs in patients with RIA-dSN MG and assessed their sensitivity in RIA-positive MG samples and their specificity. METHODS: AChR and MuSK Abs were tested in 292 serum samples from 2 Italian MG referral centers by live and fixed CBAs: 192 from patients with MG and 100 from controls. All samples had been previously assessed by RIA: 66 were AChR positive, 40 MuSK positive, and 86 dSN. All controls were negative. Two independent raters assessed the CBA results. Fixed and live CBAs were compared with the McNemar test; interrater and interlaboratory agreement were assessed with Cohen's kappa or interclass correlation coefficient (ICC), as appropriate. RESULTS: In 86 RIA-dSN samples, fixed CBA detected Abs in 10 cases (11.6%, 95% CI 5.7-20.3), whereas live CBA detected Abs in 16 (18.6%, 95% CI 11.0-28.5) (p = 0.0143). Of these sera, those positive by fixed CBA were also positive by live CBA. In addition, live CBA could detect MuSK Abs in 4 and AChR Abs in 2 samples that were negative by fixed CBA, providing an 8% (95% CI 2.9-16.6) further increase in the Ab detection rate. These results were confirmed by flow cytometry. In the RIA-positive cohort, the sensitivity for AChR Abs was 98.5% (95% CI 91.9%-99.9%) for fixed CBA and 100% (95% CI 94.6-100) for live CBA (p = 0.1573). For both assays, the sensitivity for MuSK Abs was 100% (95% CI 91.2-100), and the specificity was 100% (95% CI 96.4-100). Interrater agreement was almost perfect for live and fixed CBAs (Cohen's kappa 0.972 and 0.978, respectively), alike interlaboratory agreement. Interrater agreement for the CBA score ranged from good to excellent (ICC: 0.832-0.973). DISCUSSION: Fixed CBA represents a valuable alternative to RIA for AChR and MuSK Ab detection in patients with MG and could be considered as a first-step diagnostic test. Live CBA can be useful in the serologic evaluation of RIA- and fixed CBA-negative samples.

Immunological response after SARS-CoV-2 infection and mRNA vaccines in patients with myasthenia gravis treated with Rituximab.

In this study we employed a comprehensive immune profiling approach to determine innate and adaptive immune response to SARS-CoV-2 infection and mRNA vaccines in patients with myasthenia gravis receiving rituximab. By multicolour cytometry, dendritic and natural killer cells, B- and T-cell subsets, including T-cells producing IFN-γ stimulated with SARS-CoV-2 peptides, were analysed after infection and mRNA vaccination. In the same conditions, anti-spike antibodies and cytokines' levels were measured in sera. Despite the impaired B cell and humoral response, rituximab patients showed an intact innate, CD8 T-cell and IFN-γ specific CD4+ and CD8+ T-cell response after infection and vaccination, comparable to controls. No signs of cytokine mediated inflammatory cascade was observed. Our study provides evidence of protective immune response after SARS-CoV-2 infection and mRNA vaccines in patients with myasthenia gravis on B cell depleting therapy and highlights the need for prospective studies with larger cohorts to clarify the role of B cells in SARS-CoV-2 immune response.