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.

Clinical, immunological and molecular findings of patients with DOCK-8 deficiency from India.

DOCK8 deficiency affects various cell subsets belonging to both the innate and adaptive immune systems. Clinical diagnosis is challenging, as many cases present with severe atopic dermatitis as the only initial manifestation. Though flow cytometry helps in the presumptive diagnosis of DOCK8-deficient patients by evaluating their DOCK8 protein expression, it requires subsequent confirmation by molecular genetic analysis. Currently, haematopoietic stem cell transplantation (HSCT) is the only curative treatment option available for these patients. There is a paucity of data from India on the clinical diversity and molecular spectrum of DOCK8 deficiency. In the present study, we report the clinical, immunological and molecular findings of 17 DOCK8-deficient patients from India diagnosed over the last 5 years.

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.

CD8 + T Cells Exhibit an Exhausted Phenotype in Hemophagocytic Lymphohistiocytosis.

PURPOSE: Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory syndrome mainly caused by uncontrolled activation of antigen presenting cells and CD8 T cells. CD8 T cell exhaustion is a known phenomenon in chronic viral infections and cancer. However, the role of T cell exhaustion is not yet identified in HLH in the background of persistent inflammation. So, currently, we have characterized the CD8 T cells using flow cytometry to understand the phenomenon of exhaustion in these cells in HLH. METHODS: We have comprehensively evaluated lymphocyte subsets and characterized CD8 T cells using immunophenotypic markers like PD1, TIM3, LAG3, Ki67, Granzyme B, etc. in a cohort of 21 HLH patients. Effector cytokine secretion and degranulation by CD8 T cells are also studied. RESULTS: Our findings indicate skewed lymphocyte subsets and aberrantly activated CD8 T cells in HLH. CD8 T cells exhibit significantly increased expression of PD1, TIM3, and LAG3 prominently in primary HLH as compared to controls. PD1 + CD8 T cells express elevated levels of Granzyme B and Ki67. Moreover, CD8 T cells are hypofunctional as evidenced by significantly reduced cytokine secretion and compromised CD107a degranulation. CONCLUSION: The study has revealed that CD8 + cytotoxic T lymphocytes from HLH patients exhibited high expression of exhaustion markers with overall impaired function. To the best of our understanding, this is the first report suggesting functional exhaustion of CD8 T cells in both primary and secondary HLH. Future studies to understand the association of exhaustion with disease outcome are needed for its probable therapeutic implementation.

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.

Liposomal nanotheranostics for multimode targeted in vivo bioimaging and near-infrared light mediated cancer therapy.

Developing a nanotheranostic agent with better image resolution and high accumulation into solid tumor microenvironment is a challenging task. Herein, we established a light mediated phototriggered strategy for enhanced tumor accumulation of nanohybrids. A multifunctional liposome based nanotheranostics loaded with gold nanoparticles (AuNPs) and emissive graphene quantum dots (GQDs) were engineered named as NFGL. Further, doxorubicin hydrochloride was encapsulated in NFGL to exhibit phototriggered chemotherapy and functionalized with folic acid targeting ligands. Encapsulated agents showed imaging bimodality for in vivo tumor diagnosis due to their high contrast and emissive nature. Targeted NFGL nanohybrids demonstrated near infrared light (NIR, 750 nm) mediated tumor reduction because of generated heat and Reactive Oxygen Species (ROS). Moreover, NFGL nanohybrids exhibited remarkable ROS scavenging ability as compared to GQDs loaded liposomes validated by antitumor study. Hence, this approach and engineered system could open new direction for targeted imaging and cancer therapy.

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.