Role of Flow Cytometry in the Diagnosis of Inborn Errors of Immunity

Inborn errors of immunity (IEI) are a group of inherited heterogeneous disorders affecting the immune system characterized by increased susceptibility to infections, immune dysregulation, and lymphoproliferation. Flow cytometry (FCM) is a rapid and reliable technique for evaluation and enumeration of immune cells. It also helps in understanding the functional and signaling pathways of the immune system. Lymphocyte subset analysis is a simple and effective screening tool in suspected combined and humoral immunodeficiency patients. Qualitative phagocytic defects such as chronic granulomatous disease and leucocyte adhesion defect are easily diagnosed by FCM. Study of intracellular proteins (e.g., BTK, WASP, DOCK8), cytokine production, and signaling molecules (e.g., STAT3) by FCM is very useful but also quite challenging to establish. T and B lymphocyte interaction for normal class switching of B cells can be assessed and can help in diagnosis of combined variable immunodeficiency and hyperimmunoglobulin M syndrome. FCM is also used in posttransplant monitoring of IEI patients and also in prenatal diagnosis in suspected cases. It is also useful in validation of variants of uncertain significance obtained in exome sequencing. FCM results should always be interpreted with clinical history and, if needed, should be confirmed with molecular genetic studies before establishing the final diagnosis. Ensuring good sample quality and running parallel controls with patient samples will avoid the preanalytical and analytical errors. This review describes the applications of FCM in the diagnosis of various IEI.

Toll-like Receptor 3 Deficiency in a Child with Recurrent Infections and Diabetes Mellitus

There are more than 400 different primary immune deficiencies worldwide. Amongst them, patients with humoral immunodeficiency are more common. Most of the innate immune defects, affect the phagocytic system. There are a few cases of toll-like receptor deficiency with innate immune defects, like TLR3 mutations, which usually present with Herpes simplex encephalitis. Herein, we report a two-year old boy with TLR3 deficiency, who was presented with recurrent infections and type one diabetes mellitus.

Septic arthritis due to Mycoplasma orale in a young patient with hypogammaglobulinemia

Mycoplasma orale is an obligate intracellular bacterium usually found as a commensal in the human oral cavity. Symptomatic infections with this organism are rare, but severe disease has been described in the setting of impaired humoral immunity. Here, we describe a case in which M. orale was identified from the joint fluid of a patient with septic arthritis, splenic lesions, and agammaglobulinemia. A 15-year-old boy was admitted to the hospital with fever, progressive left knee swelling, and pain. His past medical history was significant for Burkitt’s lymphoma, the treatment of which had included rituximab 6 years earlier. M. orale was identified in the synovial fluid using 16S ribosomal RNA gene sequencing. He was also found to be hypogammaglobulinemic, and imaging revealed multiple splenic lesions. He was treated with doxycycline and intravenous immunoglobulin, which resulted in complete resolution of his arthritis and other symptoms. Mycoplasma species should be suspected in patients with humoral immunodeficiency and compatible findings.

A Patient with Kabuki Syndrome Mutation Presenting with Very Severe Aplastic Anemia

Kabuki syndrome (KS) is a rare congenital disorder commonly complicated by humoral immunodeficiency. Patients with KS present with mutation in the histone-lysine N-methyltransferase 2D (<i>KMT2D</i>) gene. Although various <i>KMT2D</i> mutations are often identified in lymphoma and leukemia, those encountered in aplastic anemia (AA) are limited. Herein, we present the case of a 45-year-old Japanese man who developed severe pancytopenia and hypogammaglobulinemia. He did not present with any evident malformations, intellectual disability, or detectable levels of autoantibodies. However, B-cell development was impaired. Therefore, a diagnosis of very severe AA due to a hypoplastic marrow, which did not respond to granulocyte colony-stimulating factor, was made. The patient received umbilical cord blood transplantation but died from a <i>Pseudomonas</i> infection before neutrophil engraftment. Trio whole-exome sequencing revealed a novel missense heterozygous mutation c.15959G &#x3e;A (p.R5320H) in exon 50 of the <i>KMT2D</i> gene. Moreover, Sanger sequencing of peripheral blood and bone marrow mononuclear cells and a skin biopsy specimen obtained from this patient identified this heterozygous mutation, suggesting that de novo mutation associated with KS occurred in the early embryonic development. Our case showed a novel association between KS mutation and adult-onset AA.

COVID-19 Infection With Humoral Immunodeficiency in Midwestern United States

Background: The coronavirus 2019 disease (COVID-19) has infected many individuals worldwide and continues to pose a significant threat to those with weakened immune systems. A paucity of data exists evaluating the clinical outcomes of patients with humoral immunodeficiencies that contract COVID-19. Objective: To describe the clinical outcomes of COVID-19 infections in patients with primary humoral immunodeficiency. Methods: We conducted a retrospective cohort review on 15 patients with a humoral immunodeficiency including Common Variable Immunodeficiency, Specific Antibody Deficiency, or unspecified hypogammaglobulinemia, who contracted COVID-19. Severity scores were determined to evaluate the clinical outcomes of these patients. Results: Of our 15-patient cohort, 33% of individuals with a humoral immunodeficiency infected with COVID-19 had a more severe disease, requiring hospitalization or resulting in death. COVID-19 mortality rate was found to be 7%. All 5 of our patients with more severe infection had at least 1 comorbidity. Conclusion: Within our cohort of humoral immunodeficient patients infected with COVID-19 we found a higher rate of severe infections and worse clinical outcomes.

SARS-CoV-2 Neutralization in Commercial Lots of Plasma-derived Immunoglobulin

Introduction: Patients suffering from primary or secondary immunodeficiency face times of increased insecurity and discomfort in the light of the raging Covid-19 pandemic, not knowing if and to what extend their comorbidities impact a potential Covid-19 course of disease. Furthermore, recently available vaccination options might not be amenable or effective for all patients of this heterogeneous population. Therefore, these patients often rely on passive immunization with plasma-derived, intravenous or subcutaneous immunoglobulin (IVIG/SCIG). Whether the ongoing Covid-19 pandemic and/or the progress in vaccination programs lead to increased and potentially protective titers in plasma-derived immunoglobulins (Ig) indicated e.g. for humoral immunodeficiency remains a pressing question for this patient population. Purpose: Here we investigated SARS-CoV-2 reactivity of US plasma-derived IVIG/SCIG products from the end of 2020 until 06/2021 as well as in convalescent plasma (CP) from 05/2020 to 08/2020. Methods: Final containers of IVIG/SCIG and CP donations were analyzed by commercial ELISA for SARS-CoV-2 S1-RBD IgG as well as microneutralization assay using a patient-derived SARS-CoV-2 (D614G) isolate. Neutralization capacities of 313 plasma single donations and 119 plasma-derived IVIG/SCIG lots were determined. Results obtained from both analytical methods were normalized against the international WHO standard. Finally, based on dense pharmacokinetic profiles of an IVIG preparation from previously published investigations, possible steady-state plasma levels of SARS-CoV-2 neutralization capacities were approximated based on currently measured anti-SARS-CoV-2 potencies in IVIG/SCIG preparations. Results: CP donations presented with a high variability with regards to anti-SARS-reactivity in ELISA as well as in neutralization testing. While approximately 50% of convalescent donations were none/low neutralizing, approximately 10% were at or above 1000 IU/ml. IVIG/SCIG lots derived from pre-pandemic plasma donations did not show neutralizing capacities of SARS-CoV-2. Lots produced between 12/2020 and 06/2021, entailing plasma donations after emergence of SARS-CoV-2 showed a rapid and constant increase in anti-SARS-CoV-2 reactivity and neutralization capacity over time. Neutralization capacity increased from a mean of 20 IU/ml in 12/2020 to 505 IU/ml in 06/2021, while lot-to-lot variability was substantial. Pharmacokinetic (PK) extrapolations based on non-compartmental superposition principles using steady-state reference profiles from previously published PK investigations on IVIG in PID, yielded potential steady-state trough plasma levels of 16 IU/ml based on the average final container concentration from 05/2021 with 216 IU/ml. Maximum extrapolated trough levels could reach 64 IU/ml based on the latest maximal final container potency tested in 06/2021. Conclusions: SARS-CoV-2 reactivity and neutralization capacity in IVIG/SCIG produced from US plasma rapidly and in part exponentially increased in the first half of 2021. The observed increase of final container potencies is likely trailing the serological status of the US donor population in terms of Covid-19 convalescence and vaccination by at least 5 months due to production lead times and should in principle continue at least until fall 2021. In summary, the data support rapidly increasing levels of SARS-COV-2 antibodies in IVIG/SCIG products implicating that a certain level of protection could be possible against COVID-19 for regularly substituted PID/SID patients. Nevertheless, more research to confirm, which plasma levels are needed for protection against SARS-CoV-2 infection of immune-compromised patients is still needed.

The 13-Valent Pneumococcal Conjugate Vaccine Elicits Serological Response and Lasting Protection in Selected Patients With Primary Humoral Immunodeficiency

BackgroundPatients with primary humoral immunodeficiency are more prone to invasive as well as recurrent pneumococcal infections. Therefore, anti-pneumococcal vaccination including the 13-valent conjugate vaccine is recommended. Nevertheless, to date, no data is available on immunogenicity of this vaccine in this population.ObjectiveTo assess the immunogenicity and the persistence of protection up to one year after a 13-valent pneumococcal conjugate vaccine in patients with primary humoral immunodeficiency.MethodsTwenty-nine patients with common variable immunodeficiency or IgG subclass deficiency were vaccinated. Immune response and immune protection at baseline as well as at one, six and twelve months after vaccination were evaluated by measuring specific IgG serum concentrations (ELISA), and opsonophagocytic activities directed against selected pneumococcal (MOPA).ResultsBy ELISA, half of the patients had protective IgG concentrations before vaccination, 35.7% showed an immune response one month after vaccination, 71.4%, 66.7% and 56.0% of the patients were protected at one, six and twelve months respectively. Conversely, by MOPA, 3.4% of the patients were protected at baseline, 10.7% showed an immune response and 28.6%, 48.2% and 33.3% were protected at one, six and twelve months respectively. IgG subclass deficiency, Ig replacement therapy and higher IgG2 concentrations at diagnosis were associated with long-term protection.ConclusionPneumococcal conjugate vaccine improves immune protection and antibodies’ functionality in a subset of patients with primary immunodeficiency. Prime-boost vaccine strategy needs to be better and individually adapted.

Haematopoietic Stem Cell Transplant for Norovirus-Induced Intestinal Failure in X-linked Agammaglobulinemia

Since the first clinical description in 1952, immunoglobulin replacement therapy remains the mainstay of treatment of patients with X-linked agammaglobulinemia (XLA). However, this therapy only replaces IgG isotype and does not compensate for the loss of Bruton tyrosine kinase in non-B-lymphocytes. Patients may still therefore develop complications despite current standard of care. Here, we describe an XLA patient with persistent chronic norovirus infection, refractory to treatment and causing intestinal failure. The patient underwent haematopoietic stem cell transplantation, curing XLA and allowed clearance of norovirus prior to humoral immunoreconstitution, suggesting non-humoral immunodeficiency in these patients.

COVID-19 Outcomes in Patients Undergoing B Cell Depletion Therapy and Those with Humoral Immunodeficiency States: A Scoping Review

Background: The role of humoral immunity has been well established in reducing infection risk and facilitating viral clearance in patients with COVID-19. However, the relationship between specific antibody responses and severity of COVID-19 is less well understood. Methods: To address this question and identify gaps in knowledge, we utilized the methodology of a scoping review to interrogate risk of infection and clinical outcomes of COVID-19 in patients with iatrogenic and inborn humoral immunodeficiency states based on existing literature. Results: Among patients with iatrogenic B-cell depletion, particularly with agents targeting CD20, our analysis found increased risk of severe COVID-19 and death across a range of underlying disease states. Among patients with humoral inborn errors of immunity with COVID-19, our synthesis found that patients with dysregulated humoral immunity, predominantly common variable immunodeficiency (CVID), may be more susceptible to severe COVID-19 than patients with humoral immunodeficiency states due to X-linked agammaglobulinemia and other miscellaneous forms of humoral immunodeficiency. There were insufficient data to appraise the risk of COVID-19 infection in both populations of patients. Conclusions: Our work identifies potentially significant predictors of COVID-19 severity in patients with humoral immunodeficiency states and highlights the need for larger studies to control for clinical and biologic confounders of disease severity.