Evaluation of a hybrid telehealth care pathway for patients with axial spondyloarthritis including self-sampling at home: results of a longitudinal proof-of-concept mixed-methods study (TeleSpactive).

Patients with axial spondyloarthritis (axSpA) require close monitoring to achieve the goal of sustained disease remission. Telehealth can facilitate continuous care while relieving scarce healthcare resources. In a mixed-methods proof-of-concept study, we investigated a hybrid telehealth care axSpA pathway in patients with stable disease over 6 months. Patients used a medical app to document disease activity (BASDAI and PtGA bi-weekly, flare questionnaire weekly). To enable a remote ASDAS-CRP (TELE-ASDAS-CRP), patients used a capillary self-sampling device at home. Monitoring results were discussed and a decision was reached via shared decision-making whether a pre-planned 3-month on-site appointment (T3) was necessary. Ten patients completed the study, and eight patients also completed additional telephone interviews. Questionnaire adherence was high; BASDAI (82.3%), flares (74.8%) and all patients successfully completed the TELE-ASDAS-CRP for the T3 evaluation. At T3, 9/10 patients were in remission or low disease activity and all patients declined the offer of an optional T3 on-site appointment. Patient acceptance of all study components was high with a net promoter score (NPS) of +50% (mean NPS 8.8 ± 1.5) for self-sampling, +70% (mean NPS 9.0 ± 1.6) for the electronic questionnaires and +90% for the T3 teleconsultation (mean NPS 9.7 ± 0.6). In interviews, patients reported benefits such as a better overview of their condition, ease of use of telehealth tools, greater autonomy, and, most importantly, travel time savings. To our knowledge, this is the first study to investigate a hybrid approach to follow-up axSpA patients including self-sampling. The positive results observed in this scalable proof-of-concept study warrant a larger confirmatory study.

Functional Relevance of CTLA4 Variants: an Upgraded Approach to Assess CTLA4-Dependent Transendocytosis by Flow Cytometry.

Variants of uncertain significance (VUS) in CTLA4 are frequently identified in patients with antibody deficiency or immune dysregulation syndromes including, but not limited to, patients with multi-organ autoimmunity and autoinflammation. However, to ascertain the diagnosis of CTLA4 insufficiency, the functional relevance of each variant needs to be determined. Currently, various assays have been proposed to assess the functionality of CTLA4 VUS, including the analysis of transendocytosis, the biological function of CTLA4 to capture CD80 molecules from antigen presenting cells. Challenges of this assay include weak fluorescence intensity of the internalized ligand, poor reproducibility, and poor performance upon analyzing thawed cells. In addition, the distinction of pathogenic from non-pathogenic variants and from wild-type CTLA4, and the classification of the different VUS according to its level of CTLA4 dysfunction, would be desirable. We developed a novel CD80-expressing cell line for the evaluation of CD80-transendocytosis and compared it to the published transendocytosis assay. Our approach showed lower inter-assay variability and better robustness regardless the type of starting material (fresh or thawed peripheral mononuclear cells). In addition, receiver operating characteristic analysis showed 100% specificity, avoiding false positive results and allowing for a clear distinction between pathogenic and non-pathogenic variants in CTLA4-variant carriers. With our transendocytosis assay, we assessed the pathogenicity of 24 distinct CTLA4 variants from patients submitted to our diagnostic unit. Significantly impaired transendocytosis was demonstrated for 17 CTLA4 variants, whereas seven variants tested normal. In conclusion, our upgraded transendocytosis assay allows a reliable assessment of newly identified variants in CTLA4.

Therapeutic options for CTLA-4 insufficiency.

BACKGROUND: Heterozygous germline mutations in cytotoxic T lymphocyte-associated antigen-4 (CTLA4) impair the immunomodulatory function of regulatory T cells. Affected individuals are prone to life-threatening autoimmune and lymphoproliferative complications. A number of therapeutic options are currently being used with variable effectiveness. OBJECTIVE: Our aim was to characterize the responsiveness of patients with CTLA-4 insufficiency to specific therapies and provide recommendations for the diagnostic workup and therapy at an organ-specific level. METHODS: Clinical features, laboratory findings, and response to treatment were reviewed retrospectively in an international cohort of 173 carriers of CTLA4 mutation. Patients were followed between 2014 and 2020 for a total of 2624 months from diagnosis. Clinical manifestations were grouped on the basis of organ-specific involvement. Medication use and response were recorded and evaluated. RESULTS: Among the 173 CTLA4 mutation carriers, 123 (71%) had been treated for immune complications. Abatacept, rituximab, sirolimus, and corticosteroids ameliorated disease severity, especially in cases of cytopenias and lymphocytic organ infiltration of the gut, lungs, and central nervous system. Immunoglobulin replacement was effective in prevention of infection. Only 4 of 16 patients (25%) with cytopenia who underwent splenectomy had a sustained clinical response. Cure was achieved with stem cell transplantation in 13 of 18 patients (72%). As a result of the aforementioned methods, organ-specific treatment pathways were developed. CONCLUSION: Systemic immunosuppressants and abatacept may provide partial control but require ongoing administration. Allogeneic hematopoietic stem cell transplantation offers a possible cure for patients with CTLA-4 insufficiency.

Pembrolizumab for treatment of progressive multifocal leukoencephalopathy in primary immunodeficiency and/or hematologic malignancy: a case series of five patients.

Progressive multifocal leukoencephalopathy is a rare opportunistic infection of the brain by John Cunningham polyomavirus in immune-compromised patients. In cases where no overt option for immune reconstitution is available [e.g., in patients with primary immunodeficiency (PID)], the disease is lethal in the majority of patients. Immune checkpoint inhibition has been applied in recent years with mixed outcomes. We present four novel patients and the follow-up of a previously published patient suffering from progressive multifocal leukoencephalopathy (PML) due to PID and/or hematologic malignancy who were treated with the immune checkpoint inhibitor pembrolizumab. In two patients with PID, symptoms improved and stabilized. One patient died because of worsening PML another of intracranial hemorrhage which was unrelated to PML or its treatment with pembrolizumab. The fifth patient suffered from PID and died of a pre-existing immune dysregulation, possibly exacerbated by pembrolizumab. The long-term follow-up of the first patient provides support for therapeutic decisions during this therapy and is the longest published clinical course of a patient with checkpoint inhibition for PML. We conclude that pembrolizumab can control PML symptoms long term in a subgroup of patients with PID, in our cases for 21 and 36 months. However, therapy must be started early because symptoms are only partially reversible. In light of severe adverse events, application of pembrolizumab is only justified if the prognosis for the individual patient is very poor.

Genetic Analysis of a Cohort of 275 Patients with Hyper-IgE Syndromes and/or Chronic Mucocutaneous Candidiasis.

Hyper-IgE syndromes and chronic mucocutaneous candidiasis constitute rare primary immunodeficiency syndromes with an overlapping clinical phenotype. In recent years, a growing number of underlying genetic defects have been identified. To characterize the underlying genetic defects in a large international cohort of 275 patients, of whom 211 had been clinically diagnosed with hyper-IgE syndrome and 64 with chronic mucocutaneous candidiasis, targeted panel sequencing was performed, relying on Agilent HaloPlex and Illumina MiSeq technologies. The targeted panel sequencing approach allowed us to identify 87 (32 novel and 55 previously described) mutations in 78 patients, which generated a diagnostic success rate of 28.4%. Specifically, mutations in DOCK8 (26 patients), STAT3 (21), STAT1 (15), CARD9 (6), AIRE (3), IL17RA (2), SPINK5 (3), ZNF341 (2), CARMIL2/RLTPR (1), IL12RB1 (1), and WAS (1) have been detected. The most common clinical findings in this cohort were elevated IgE (81.5%), eczema (71.7%), and eosinophilia (62.9%). Regarding infections, 54.7% of patients had a history of radiologically proven pneumonia, and 28.3% have had other serious infections. History of fungal infection was noted in 53% of cases and skin abscesses in 52.9%. Skeletal or dental abnormalities were observed in 46.2% of patients with a characteristic face being the most commonly reported feature (23.1%), followed by retained primary teeth in 18.9% of patients. Targeted panel sequencing provides a cost-effective first-line genetic screening method which allows for the identification of mutations also in patients with atypical clinical presentations and should be routinely implemented in referral centers.

Establishing the Molecular Diagnoses in a Cohort of 291 Patients With Predominantly Antibody Deficiency by Targeted Next-Generation Sequencing: Experience From a Monocentric Study.

Predominantly antibody deficiencies (PAD) are a heterogeneous group of disorders characterized by dysfunctional antibody production, low immunoglobulin levels in serum and impaired vaccine responses. The clinical picture is variable, ranging from mild symptoms to severe complications, which may include autoimmunity, gastrointestinal disease, allergy, and malignancies. If left untreated, PAD patients are at risk of enduring disease progression, irreversible organ damage, and reduced life expectancy. A timely diagnosis has been shown to significantly improve disease prognosis. Here, we report on our experience using targeted gene panel sequencing by employing Agilent's HaloPlex or SureSelect and Illumina's MiSeq technologies in a cohort of 291 individuals who presented with low or absent immunoglobulin levels in combination with or without other clinical features. In total, we have detected over 57 novel or previously reported relevant mutations in ADA, ADA2, BTK, CTLA4, LRBA, NFKB1, NFKB2, PIK3CD, STAT3, and TNFRSF13B. Overall, a genetic diagnosis could be made in 24.7% of the investigated patients. The percentage of coverage for the targeted regions ranged from 90% to 98% in this study. Moreover, functional assays were performed on a defined group of the patients carrying candidate variants in CTLA4, LRBA, NFKB1 and BTK, which confirmed their deleterious effect on protein expression and/or function. This study reiterates that the immunological heterogeneity of predominantly antibody deficiencies may have a diverse genetic origin, although certain clinical features may hint towards a specific group of defects. Employing targeted sequencing panels proves to be a very time- and cost-efficient, yet reliable, method for the establishment of a genetic diagnosis in individuals with PAD. However, in case of negative panel results, or if functional testing reveals inconspicuous observations in patients with a clear indication for genetic testing, further work-up including whole exome or whole genome sequencing should be considered.

Clinical Phenotypes and Immunological Characteristics of 18 Egyptian LRBA Deficiency Patients.

LPS-responsive beige-like anchor (LRBA) deficiency is an autosomal recessive primary immunodeficiency disorder, OMIM (#614700). LRBA deficiency patients suffer from variable manifestations including recurrent infections, immune dysregulation, autoimmunity, cytopenias, and enteropathy. This study describes different clinical phenotypes and immunological characteristics of 18 LRBA deficiency patients diagnosed from Egypt. T and B lymphocyte subpopulations, LRBA, and cytotoxic T lymphocyte-associated protein 4 (CTLA4) expression were evaluated in resting and stimulated T cells using flow cytometry. Next-generation sequencing was used to identify mutations in the LRBA gene. LRBA deficiency patients had significantly lower B cells and increased percentage of memory T cells. CTLA4 levels were lower in LRBA-deficient T regulatory cells in comparison to healthy donors at resting conditions and significantly increased upon stimulation of T cells. We identified 11 novel mutations in LRBA gene ranging from large deletions to point mutations. Finally, we were able to differentiate LRBA-deficient patients from healthy control and common variable immunodeficiency patients using a simple flow cytometry test performed on whole blood and without need to prior stimulation. LRBA deficiency has heterogeneous phenotypes with poor phenotype-genotype correlation since the same mutation may manifest differently even within the same family. Low LRBA expression, low numbers of B cells, increased numbers of memory T cells, and defective CTLA4 expression (which increase to normal level upon T cell stimulation) are useful laboratory tests to establish the diagnosis of LRBA deficiency. Screening of the siblings of affected patients is very important as patients may be asymptomatic at the beginning of the disease course.

Clinical and Immunological Phenotype of Patients With Primary Immunodeficiency Due to Damaging Mutations in NFKB2.

Non-canonical NF-κB-pathway signaling is integral in immunoregulation. Heterozygous mutations in NFKB2 have recently been established as a molecular cause of common variable immunodeficiency (CVID) and DAVID-syndrome, a rare condition combining deficiency of anterior pituitary hormone with CVID. Here, we investigate 15 previously unreported patients with primary immunodeficiency (PID) from eleven unrelated families with heterozygous NFKB2-mutations including eight patients with the common p.Arg853* nonsense mutation and five patients harboring unique novel C-terminal truncating mutations. In addition, we describe the clinical phenotype of two patients with proximal truncating mutations. Cohort analysis extended to all 35 previously published NFKB2-cases revealed occurrence of early-onset PID in 46/50 patients (mean age of onset 5.9 years, median 4.0 years). ACTH-deficiency occurred in 44%. Three mutation carriers have deceased, four developed malignancies. Only two mutation carriers were clinically asymptomatic. In contrast to typical CVID, most patients suffered from early-onset and severe disease manifestations, including clinical signs of T cell dysfunction e.g., chronic-viral or opportunistic infections. In addition, 80% of patients suffered from (predominately T cell mediated) autoimmune (AI) phenomena (alopecia > various lymphocytic organ-infiltration > diarrhea > arthritis > AI-cytopenia). Unlike in other forms of CVID, auto-antibodies or lymphoproliferation were not common hallmarks of disease. Immunophenotyping showed largely normal or even increased quantities of naïve and memory CD4+ or CD8+ T-cells and normal T-cell proliferation. NK-cell number and function were also normal. In contrast, impaired B-cell differentiation and hypogammaglobinemia were consistent features of NFKB2-associated disease. In addition, an array of lymphocyte subpopulations, such as regulatory T cell, Th17-, cTFH-, NKT-, and MAIT-cell numbers were decreased. We conclude that heterozygous damaging mutations in NFKB2 represent a distinct PID entity exceeding the usual clinical spectrum of CVID. Impairment of the non-canonical NF-κB pathways affects function and differentiation of numerous lymphocyte-subpopulations and thus causes a heterogeneous, more severe form of PID phenotype with early-onset. Further characteristic features are multifaceted, primarily T cell-mediated autoimmunity, such as alopecia, lymphocytic organ infiltration, and in addition frequently ACTH-deficiency.

ZNF341 controls STAT3 expression and thereby immunocompetence.

Signal transducer and activator of transcription 3 (STAT3) is a central regulator of immune homeostasis. STAT3 levels are strictly controlled, and STAT3 impairment contributes to several diseases including the monogenic autosomal-dominant hyper-immunoglobulin E (IgE) syndrome (AD-HIES). We investigated patients of four consanguineous families with an autosomal-recessive disorder resembling the phenotype of AD-HIES, with symptoms of immunodeficiency, recurrent infections, skeletal abnormalities, and elevated IgE. Patients presented with reduced STAT3 expression and diminished T helper 17 cell numbers, in absence of STAT3 mutations. We identified two distinct homozygous nonsense mutations in ZNF341, which encodes a zinc finger transcription factor. Wild-type ZNF341 bound to and activated the STAT3 promoter, whereas the mutant variants showed impaired transcriptional activation, partly due to nuclear translocation failure. In summary, nonsense mutations in ZNF341 account for the STAT3-like phenotype in four autosomal-recessive kindreds. Thus, ZNF341 is a previously unrecognized regulator of immune homeostasis.

Targeted Gene Panel Sequencing for Early-onset Inflammatory Bowel Disease and Chronic Diarrhea.

BACKGROUND: In contrast to adult-onset inflammatory bowel disease (IBD), where many genetic loci have been shown to be involved in complex disease etiology, early-onset IBD (eoIBD) and associated syndromes can sometimes present as monogenic conditions. As a result, the clinical phenotype and ideal disease management in these patients often differ from those in adult-onset IBD. However, due to high costs and the complexity of data analysis, high-throughput screening for genetic causes has not yet become a standard part of the diagnostic work-up of eoIBD patients. METHODS: We selected 28 genes of interest associated with monogenic IBD and performed targeted panel sequencing in 71 patients diagnosed with eoIBD or early-onset chronic diarrhea to detect causative variants. We compared these results to whole-exome sequencing (WES) data available for 25 of these patients. RESULTS: Target coverage was significantly higher in the targeted gene panel approach compared with WES, whereas the cost of the panel was considerably lower (approximately 25% of WES). Disease-causing variants affecting protein function were identified in 5 patients (7%), located in genes of the IL10 signaling pathway (3), WAS (1), and DKC1 (1). The functional effects of 8 candidate variants in 5 additional patients (7%) are under further investigation. WES did not identify additional causative mutations in 25 patients. CONCLUSIONS: Targeted gene panel sequencing is a fast and effective screening method for monogenic causes of eoIBD that should be routinely established in national referral centers.

Screening of 181 Patients With Antibody Deficiency for Deficiency of Adenosine Deaminase 2 Sheds New Light on the Disease in Adulthood.

OBJECTIVE: We aimed to test the relevance of deficiency of adenosine deaminase 2 (DADA2) in patients with antibody deficiency and describe the clinical picture of the disease in adulthood. METHODS: We screened for DADA2 in a cohort of 181 patients with antibody deficiency with or without vascular lesions using next-generation sequencing and targeted Sanger sequencing. All mutations were confirmed by determining the ADA2 enzymatic activity levels in dried plasma spots. Clinical data and laboratory values were collected in a standardized format. RESULTS: Following the diagnosis of 2 siblings in the index family, we identified 9 additional affected patients with compound heterozygous or homozygous CECR1 mutations, containing 6 novel and 4 previously published mutations. The patients' age at evaluation ranged from 13 to 51 years, with a median age of 22 years. Clinically, we saw a broad phenotype, ranging from isolated antibody deficiency to recurrent strokes. All but 1 patient had low numbers of memory B cells. Moreover, B cell function seemed to correlate with inflammation. CONCLUSION: Taken together, our findings indicate that DADA2 presents not only with vasculopathy but also with an immunodeficiency of the B cell compartment. Therefore, patients with antibody deficiency should be screened for DADA2. Anti-tumor necrosis factor treatment might improve immunologic features over time and might be considered in patients without vascular manifestations but with elevated inflammation markers. Conservative management has so far proven to be the choice for our less severely affected adolescent and adult DADA2 patients; however, in patients with severe cytopenias and bone marrow failure, hematopoietic stem cell transplantation should be considered.

Validación de la técnica de dihidrorodamina 123 para el diagnóstico de la enfermedad granulomatosa crónica en Colombia / Validation of the dyhidrorhodamine test for the diagnosis of chronic granulomatous disease in Colombia

Introducción: La enfermedad granulomatosa crónica (EGC) es una inmunodeficiencia primaria por defectos en la explosión respiratoria de los fagocitos cuyas manifestaciones clínicas más frecuentes son los granulomas y las infecciones recurrentes por gérmenes encapsulados. Objetivo Estandarizar en Colombia la técnica de dihidrorodamina (DHR) para diagnóstico de EGC con el propósito de evaluar la explosión respiratoria en neutrófilos y monocitos de sangre periférica (SP) humana después de ser activados in vitro. Métodos Los leucocitos de SP de 10 individuos sanos fueron activados utilizando varias concentraciones de PMA, evaluando la explosión respiratoria por citometría de flujo. Adicionalmente, se obtuvieron los patrones de oxidación de la DHR de estas células en EGC ligada al X, portadoras de este defecto, y en EGC autosómica recesiva. Resultados Las mejores concentraciones de PMA para evaluar el estallido respiratorio tanto en neutrófilos como en monocitos se encontraron entre 0,2 y 5 μg/ml. Se establecieron parámetros de normalidad de la prueba en neutrófilos en nuestra población. Adicionalmente, los patrones de oxidación de la DHR en monocitos no siempre fueron los mismos a aquellos de los neutrófilos. Conclusiones La técnica de DHR por citometría de flujo es un método de cribado que identifica con facilidad, sensibilidad y a bajo costo los diferentes fenotipos de la EGC. Sin embargo, se deben establecer en cada laboratorio los parámetros de normalidad que permiten realizar con certeza la caracterización inicial de esta condición y evaluar diferentes tipos celulares teniendo en cuenta que la expresividad del defecto puede ser específica de ciertas poblaciones celulares (AU)

Introduction: Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by defects in the respiratory burst of phagocytes. Affected patients often suffer from granulomas and recurrent infections, mainly due to encapsulated bacteria. Aim: To standardize the dihydrorhodamine test (DHR) in Colombia used for the diagnosis of CGD by evaluating the respiratory burst in human blood neutrophils and monocytes after in vitro activation. Methods: Phagocyte respiratory burst in peripheral blood samples from 10 healthy controls was evaluated by flow cytometry after leukocyte activation with several concentrations of phorbol myristate acetate (PMA). The different oxidation patterns of DHR in X-linked or autosomal recessive CGD were also obtained. Results: The most suitable concentrations of PMA for the evaluation of the respiratory burst in peripheral blood were 0.2 to 5g/ml. Reference values for this test in neutrophils from our population were established. It was shown that the oxidation patterns of DHR in monocytes were not always identical to those observed in neutrophils. Conclusion: The evaluation of DHR oxidation by flow cytometry is a screening method that easily identifies the different phenotypes of CGD, with good sensitivity and at a lower cost. However, it is crucial that every laboratory establishes its own normal range for this test, in order to achieve the accurate characterization of this condition. DHR oxidation patterns may be also evaluated in different blood cells, since cell type-specific defects have also been reported (AU)