Diagnosis and Management of Infections in Patients with Mendelian Susceptibility to Mycobacterial Disease.

The diagnosis and treatment of patients with mendelian susceptibility to mycobacterial disease (MSMD) pose consistent challenges due to the diverse infection spectrum observed in this population. Common clinical manifestations include Bacillus Calmette-Guérin vaccine (BCG) complications in countries where routine BCG vaccination is practiced, while in non-BCG-vaccinating countries, Non-Tuberculous Mycobacteria (NTM) is prevalent. In tuberculosis-endemic regions, Mycobacterium tuberculosis (MTB) has a high prevalence, along with other intracellular organisms. Isolating these organisms presents a significant challenge, and treatment is often initiated without confirming the specific species. This review primarily focuses on the methods and challenges associated with diagnosing and treating MSMD patients.

Normative data for paediatric lymphocyte subsets: A pilot study from western India.

Background & objectives: Accurate diagnosis of immunodeficiencies requires a critical comparison of values with age-matched controls. In India, the existing reference values for rare lymphocyte subsets are currently not available and we rely on the data originating from other countries for the interpretation of the results. Furthermore, there is limited information on normal variation for these rare-subset parameters in Indian children. So, this study aimed to establish normative values for clinically important lymphocyte subsets in Indian children at different age groups. Methods: 148 children aged ≥16 yr were enrolled in this study. The study population included 61 per cent males and 39 per cent females and was divided into the following groups: cord blood (n=18), 0-6 months (n=9), 6-12 months (n=13), 1-2 yr (n=19), 2-5 yr (n=27), 5-10 yr (n=25) and 10-16 yr (n=37). The absolute and relative percentage of lymphocytes, T, B, natural killer cell, along with activated, naïve and memory subsets, was determined by flow cytometry. Results: Median values and the 10th and 90th percentiles were obtained for 34 lymphocyte sub-populations. The T and B naïve compartments showed a decreasing trend, whereas memory cells showed an increase with age. The activated T cell subset shows an increasing pattern up to one year and then declines gradually. Double negative T cells are relatively stable. TCRgd+T cell percentage increases with age. Interpretation & conclusions: This single-centre pilot study provides preliminary data that justifies the need for future large-scale multi centric studies to generate a reference range for interpreting extended immunophenotyping profiles in the paediatric age group, making it possible for clinicians to assess the immunological status in inborn errors of immunity, infectious and autoimmune diseases.

A Clinical Conundrum with Diagnostic and Therapeutic Challenge: a Tale of Two Disorders in One Case.

Living organisms are exposed to exogenous and endogenous agents that affect genomic integrity by creating DNA double strand breaks (DSBs). These breaks are repaired by DNA repair proteins to maintain homeostasis. Defects in DNA repair pathways also affect lymphocyte development and maturation, as DSB sites are critical intermediates for rearrangements required for V(D)J recombination. Recent classifications for inborn errors of immunity (IEIs) have listed DNA repair defect genes in a separate group, which suggests the importance of these genes for adaptive and innate immunity. We report an interesting case of a young female (index P1) with mutations in two different genes, DCLRE1C and FANCA, involved in DNA repair pathways. She presented with clinical manifestations attributed to both defects. With the advent of NGS, more than one defect is increasingly identified in patients with IEIs. Familial segregation studies and appropriate functional assays help ascertain the pathogenicity of these mutations and provide appropriate management and genetic counseling.

Major Histocompatibility Complex (MHC) Class II Deficiency- A Case of Primary Immunodeficiency Disorder.

Major Histocompatibility Complex (MHC) Class II deficiency is a rare autosomal recessive primary immunodeficiency caused by mutations in regulatory genes of MHC Class II proteins. Clinical manifestations include respiratory/gastrointestinal infections, failure-to-thrive, septicemia and early death. A 9-mo-old-girl presented with repeated episodes of pneumonia requiring hospitalization and ventilator support since the last 5 mo. Examination revealed absent tonsils, sparse scalp-hair, seborrhea and firm hepato-splenomegaly. Radiograph showed absence of thymic shadow with diffuse pulmonary infiltrates. CT scan showed multiple bilateral ground glass pulmonary opacities with patchy consolidation. Primary immunodeficiency disorder was suspected in view of repeated pulmonary infections, failure to thrive and suggestive family history. Lymphocyte subset assay revealed lymphocytopenia and HLA typing showed absence of HLA-DR expression on B cells suggestive of MHC Class II deficiency. Targeted gene panel detected a homozygous mutation in the RFX-5 gene (RFX5: c.848_849del:p.R283Tfs*6;Homozygous). Though this patient succumbed, parents have been counseled regarding need for prenatal diagnosis.

Targeted next-generation sequencing revealed a novel homozygous mutation in the LRBA gene causes severe haemolysis associated with Inborn Errors of Immunity in an Indian family.

OBJECTIVES: LPS-responsive beige-like anchor protein (LRBA) deficiency abolishes LRBA protein expression due to biallelic mutations in the LRBA gene that lead to autoimmune manifestations, inflammatory bowel disease, hypogammaglobulinemia in early stages, and variable clinical manifestations. MATERIALS AND METHODS: Mutational analysis of the LRBA gene was performed in Indian patients using targeted Next Generation Sequencing (t-NGS) and confirmed by Sanger sequencing using specific primers of exons 53. Then, bioinformatics analysis and protein modeling for the novel founded mutations were also performed. The genotype, phenotype correlation was done according to the molecular findings and clinical features. RESULTS: We report an unusual case of a female patient born of a consanguineous marriage, presented with severe anaemia and jaundice with a history of multiple blood transfusions of unknown cause up to the age of 5 yrs. She had hepatosplenomegaly with recurrent viral and bacterial infections. Tests for hemoglobinopathies, enzymopathies, and hereditary spherocytosis were within the normal limits. The t-NGS revealed a novel homozygous missense variation in exon 53 of the LRBA gene (chr4:151231464C > T; c.7799G > A) (p.C2600Y), and the parents were heterozygous. The further immunological analysis is suggestive of hypogammaglobulinaemia and autoimmune haemolytic anaemia. The bioinformatics tools are suggestive of deleterious and disease-causing variants. CONCLUSION: This study concludes the importance of a timely decision of targeted exome sequencing for the molecular diagnostic tool of unexplained haemolytic anaemia with heterogeneous clinical phenotypes.

Serosurvey for Health-Care Workers Provides Supportive Evidence for the Effectiveness of Hydroxychloroquine Prophylaxis against SARS-CoV-2 Infection.

BACKGROUND: The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic has resulted in occupational exposure among Healthcare Workers (HCWs) and a high risk of nosocomial transmission. Asymptomatic infection and transmission of infection before the development of symptoms are well-recognized factors contributing to the spread of infection. We conducted a cross-sectional observational study to understand the seroprevalence of SARS-CoV-2 infection among HCWs and to verify the appropriateness of infection control measures, particularly Hydroxychloroquine (HCQ) prophylaxis. METHODS: A cross-sectional sero-surveillance study was conducted among 500 HCWs in Dombivli and surrounding Mumbai Metropolitan area (Maharashtra, India) between 21st July and 3rd August 2020. The vulnerability of the study participants to SARS-CoV-2 infection was ascertained through a history of (i) involvement in direct care, (ii) exposure to aerosol-generating procedures, (iii) co-morbidities, (iv) Personal Protective Equipment (PPE) use, and (v) HCQ prophylaxis. SARS-CoV-2 IgG antibodies were tested using COVID KAVACH anti-SARS-CoV-2 IgG antibody detection enzyme-linked immunosorbent assay (ELISA) from Zydus Cadila. A systematic analysis of the correlation between the development of antibodies and factors affecting vulnerability to infection was performed. RESULTS: The overall SARS-CoV-2 seroprevalence in the study population was 11%. Providing direct care to COVID-19 patients (Adjusted OR 16.4, 95% CI 3.3-126.9, p = 0.002) for long hours and irregular use of PPE (Adjusted OR 3.78, 95% CI 1.1-11.9, p = 0.02) were associated with an increased incidence of seropositivity. Prophylaxis with HCQ may have a role in reducing the vulnerability to infection as depicted by univariate and multivariate analysis (Adjusted OR 0.55, 95% CI 0.3-0.9, p = 0.047). It was also noted that those not on HCQ prophylaxis were threefold more prone to infection and developed severe disease as compared to those on HCQ prophylaxis. CONCLUSION: Prophylaxis with HCQ may have a role in mitigating the incidence and severity of SARS-CoV-2 infection. Although vaccination is the most robust strategy to safeguard against COVID-19, it will be months before vaccination percolates to the masses. In the face of the second wave of COVID-19, the use of HCQ prophylaxis in combination with use of face-masks regularly may be considered as a cost-effective measure for population dense areas like urban slums where social distancing is not possible.

A case of disseminated subcutaneous phaeohyphomycosis caused by Exserohilum rostratum with CARD9 mutation.

Phaeohypomycosis is a rare cutaneous and subcutaneous fungal infection caused by dematiaceous fungi. They have a widespread global distribution occasionally affecting humans. A 26-year-old woman presented with multiple skin lesions over her face and extremities for last 7 years, unresponsive to systemic amphotericin B and itraconazole. Further investigations revealed CARD9 mutation and phaeohyphomycosis caused by the pigmented fungus Exserohilum rosatratum. Lesions subsequently improved with oral flucytosine and itraconazole.

Approach to genetic diagnosis of inborn errors of immunity through next-generation sequencing.

Patients with inborn errors of immunity (IEI) present with a heterogeneous clinical and immunological phenotype, therefore a correct molecular diagnosis is crucial for the classification and subsequent therapeutic management. On the other hand, IEI are a group of rare congenital diseases with highly diverse features and, in most cases, an as yet unknown genetic etiology. Next generation sequencing has facilitated genetic examinations of rare inherited disorders during the recent years, thus allowing a suitable molecular diagnosis in the IEI patients. This review aimed to investigate the current findings about these techniques in the field of IEI, suggesting an efficient stepwise approach to molecular diagnosis of inborn errors of immunity.

Clinical and Molecular Findings in Mendelian Susceptibility to Mycobacterial Diseases: Experience From India.

Mendelian Susceptibility to Mycobacterial diseases (MSMD) are a group of innate immune defects with more than 17 genes and 32 clinical phenotypes identified. Defects in the IFN-γ mediated immunity lead to an increased susceptibility to intracellular pathogens like mycobacteria including attenuated Mycobacterium bovis-Bacillus Calmette-Guérin (BCG) vaccine strains and non-tuberculous environmental mycobacteria (NTM), Salmonella, fungi, parasites like Leishmania and some viruses, in otherwise healthy individuals. Mutations in the IL12RB1 gene are the commonest genetic defects identified. This retrospective study reports the clinical, immunological, and molecular characteristics of a cohort of 55 MSMD patients from 10 centers across India. Mycobacterial infection was confirmed by GeneXpert, Histopathology, and acid fast bacilli staining. Immunological workup included lymphocyte subset analysis, Nitro blue tetrazolium (NBT) test, immunoglobulin levels, and flow-cytometric evaluation of the IFN-γ mediated immunity. Genetic analysis was done by next generation sequencing (NGS). Disseminated BCG-osis was the commonest presenting manifestation (82%) with a median age of presentation of 6 months due to the practice of BCG vaccination at birth. This was followed by infection with Salmonella and non-typhi Salmonella (13%), Cytomegalovirus (CMV) (11%), Candida (7%), NTM (4%), and Histoplasma (2%). Thirty-six percent of patients in cohort were infected by more than one organism. This study is the largest cohort of MSMD patients reported from India to the best of our knowledge and we highlight the importance of work up for IL-12/IL-23/ISG15/IFN-γ circuit in all patients with BCG-osis and suspected MSMD irrespective of age.

The clinical efficacy of Rituximab administration in autoimmunity disorders, primary immunodeficiency diseases and malignancies.

Rituximab (RTX), as a monoclonal antibody-based immunotherapeutic intervention targeting CD20 on B cells, has proven efficacy in the treatment of patients with some immune-mediated diseases. In the present review, we provided information on the immunobiological mechanisms of signaling for RTX and its clinical applications, according to the immune-pathophysiology involved in the microenvironment of multiple diseases. We highlighted combination therapy, dose schedules, and laboratory monitoring, as well as the associated common and rare side effects to avoid. We also discussed the efficacy and safety of RTX-based therapeutic strategies and whether RTX therapy can be used as a promising treatment regimen for autoimmune diseases, primary immunodeficiency diseases, and malignancies. Our review highlights and supports the importance of collaboration between basic medical researchers and clinical specialists when considering the use of RTX in the treatment of various immune-mediated disorders.

The Spectrum of Clinical, Immunological, and Molecular Findings in Familial Hemophagocytic Lymphohistiocytosis: Experience From India.

Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of immune dysregulation characterized by hyperactivation of the immune system, excessive cytokine secretion and severe systemic inflammation. HLH is classified as familial (FHL) when associated with mutations in PRF1, UNC13D, STX11, and STXBP2 genes. There is limited information available about the clinical and mutational spectrum of FHL patients in Indian population. This study is a retrospective analysis of 101 molecularly characterized FHL patients over the last 10 years from 20 different referral centers in India. FHL2 and FHL3 together accounted for 84% of cases of FHL in our cohort. Patients belonging to different FHL subtypes were indistinguishable based on clinical and biochemical parameters. However, flow cytometry-based assays viz. perforin expression and degranulation assay were found to be specific and sensitive in diagnosis and classification of FHL patients. Molecular characterization of respective genes revealed 76 different disease-causing mutations including 39 (51%) novel mutations in PRF1, UNC13D, STX11, and STXBP2 genes. Overall, survival was poor (28%) irrespective of the age of onset or the type of mutation in our cohort. Altogether, this article sheds light on the current scenario of FHL in India. Our data reveal a wide genetic heterogeneity of FHL in the Indian population and confirms the poor prognosis of FHL. This study also emphasizes that though mutational analysis is important for diagnostic confirmation of FHL, flow cytometry based assays help significantly in rapid diagnosis and functional validation of novel variants identified.

Chalazia, A Late Manifestation of Primary Immunodeficiency Disorders.

PURPOSE: To call attention to the central clues for primary immunodeficiency in the cases reported by María Nieves-Moreno et. al where chalazia in the reported cases is undoubtedly an important clue but is a late clinical manifestation.

Spectrum of Inborn errors of immunity in a cohort of 90 patients presenting with complications to BCG vaccination in India.

World Health Organisation recommends the practice of BCG vaccination at birth in countries which have a high incidence of tuberculosis and/or high leprosy burden. The BCG vaccination is considered safe for a competent immune system. However, in children with weakened immune systems cause of which can be primary or secondary, the vaccine may lead to side effects which can be localized or disseminated. In this study, we report a spectrum of inborn errors of immunity (IEI) commonly referred to as primary immunodeficiency disorders (PIDs) diagnosed in a large cohort of patients presenting with complications to BCG vaccination from India. Retrospective data analysis of patients referred to ICMR- National Institute of Immunohematology (ICMR-NIIH) for IEI workup between 2007 and 2019 was done. IEI was identified in n = 52/90 (57.7%) patients presenting with BCG complications. Of these, n = 13(14.4%) patients were diagnosed with severe combined immune deficiency, n = 15(16.7%) with chronic granulomatous disease, n = 19(21.1%) with Inborn errors of IFN-γ immunity, n = 4(4.4%) with Combined immunodeficiency and n = 1(1.1%) with Leucocyte Adhesion Deficiency type1. Majority of cases with BCGosis (88%) had an underlying IEI. This study strongly highlights the need for evaluation of patients with BCG complications for underlying IEI. While disseminated BCGosis strongly predicts underlying IEI, even localized persistent adenitis may be a warning sign of underlying IEI. It is also strongly recommended to record a family history of previous sibling death prior to administration of this live vaccine and deferring live vaccine till the diagnosis of IEI is ruled out in cases with a positive family history.

Prenatal Diagnosis for Primary Immunodeficiency Disorders-An Overview of the Indian Scenario.

Prenatal Diagnosis (PND) forms an important part of primary preventive management for families having a child affected with primary immunodeficiency. Although individually sparse, collectively this group of genetic disorders represents a significant burden of disease. This paper discusses the prenatal services available for affected families at various centers across the country and the challenges and ethical considerations associated with genetic counseling. Mutation detection in the index case and analysis of chorionic villous sampling or amniocentesis remain the preferred procedures for PND and phenotypic analysis of cordocentesis sample is reserved for families with well-characterized index case seeking PND in the latter part of the second trimester of pregnancy. A total of 112 families were provided PND services in the last decade and the presence of an affected fetus was confirmed in 32 families. Post-test genetic counseling enabled the affected families to make an informed decision about the current pregnancy.

2019 Update on Primary Immunodeficiency Disorders by the International Union of Immunological Societies.

International Union of Immunological Societies working group recently updated the human inborn errors of immunity. This classification includes 65 new disorders that have been added since the last classification in 2018. This article highlights the important aspects of new classification for the benefit of general pediatricians.

Clinical and Genetic Spectrum of a Large Cohort of Patients With Leukocyte Adhesion Deficiency Type 1 and 3: A Multicentric Study From India.

Leukocyte adhesion deficiency (LAD) syndrome is a group of inborn errors of immunity characterized by a defect in the cascade of the activation and adhesion leading to the failure of leukocyte to migrate to the site of tissue injury. Three different types of LAD have been described. The most common subtype is LAD type 1 (LAD1) caused due to defects in the ITGß2 gene. LAD type 2 (LAD2) is caused by mutations in the SLC35C1 gene leading to a generalized loss of expression of fucosylated glycans on the cell surface and LAD type 3 (LAD3) is caused by mutations in the FERMT3 gene resulting in platelet function defects along with immunodeficiency. There is a paucity of data available from India on LAD syndromes. The present study is a retrospective analysis of patients with LAD collated from 28 different centers across India. For LAD1, the diagnosis was based on clinical features and flow cytometric expression of CD18 on peripheral blood leukocytes and molecular confirmation by Sanger sequencing. For patients with LAD3 diagnosis was largely based on clinical manifestations and identification of the pathogenic mutation in the FERMT3 gene by next-generation Sequencing. Of the total 132 cases diagnosed with LAD, 127 were LAD1 and 5 were LAD3. The majority of our patients (83%) had CD18 expression less than 2% on neutrophils (LAD1°) and presented within the first three months of life with omphalitis, skin and soft tissue infections, delayed umbilical cord detachment, otitis media, and sepsis. The patients with CD18 expression of more than 30% (LAD1+) presented later in life with skin ulcers being the commonest manifestation. Bleeding manifestations were common in patients with LAD3. Persistent neutrophilic leukocytosis was the characteristic finding in all patients. 35 novel mutations were detected in the ITGß2 gene, and 4 novel mutations were detected in the FERMT3 gene. The study thus presents one of the largest cohorts of patients from India with LAD, focusing on clinical features, immunological characteristics, and molecular spectrum.