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.

Poliovirus surveillance in patients with primary immunodeficiencies, India.

Individuals with primary immunodeficiencies who are infected with vaccine-derived polioviruses may continue to shed poliovirus for months and go undetected by surveillance programmes of acute flaccid paralysis. These patients therefore pose a risk of initiating poliovirus outbreaks that jeopardize efforts towards global polio eradication. To identify these individuals, we designed a study protocol for the establishment of a network for surveillance of immunodeficiency-related vaccine-derived poliovirus in India. In the first step we identified recognized centres in India that could diagnose and enrol patients with primary immunodeficiency disorder into the study. Stool sample collection from study sites, culture, isolation, characterization of enteroviruses and reporting to study sites was carried out at the National Institute of Virology Mumbai Unit, as per the WHO national polio surveillance project protocol. In the first phase of the study from January 2020 to December 2021, we implemented the protocol at seven study sites at different medical institutes to determine the proportion of poliovirus infections in primary immunodeficiency disorder patients of India. We later expanded the study by including an additional 14 medical institutes across the country in the second phase running from January 2022 to December 2023. We believe this study protocol will help other countries to initiate immunodeficiency-related vaccine-derived poliovirus surveillance to identify and follow up patients who are long-term excretors of vaccine-derived poliovirus. Integration of immunodeficiency-related poliovirus surveillance with acute flaccid paralysis surveillance of the existing poliovirus network will enhance continuous screening of patients with primary immunodeficiency disorder in the future.

Certains individus qui présentent des immunodéficiences primaires et sont infectés par des poliovirus dérivés d'une souche vaccinale pourraient continuer à excréter le poliovirus pendant des mois sans que ce dernier ne soit détecté par le biais d'une surveillance de la paralysie flasque aiguë. Ces patients risquent donc de déclencher des épidémies de poliovirus qui mettent en péril les efforts visant à éradiquer la poliomyélite dans le monde. En vue d'identifier ces individus, nous avons élaboré un protocole d'étude pour établir, en Inde, un réseau de surveillance du poliovirus d'origine vaccinale lié à une immunodéficience. Au cours de la première étape, nous avons repéré des centres reconnus dans le pays, capables de diagnostiquer des patients atteints d'un syndrome d'immunodéficience primaire et de les recruter dans le cadre de l'étude. Le prélèvement des échantillons de selles auprès des sites participant à l'étude, la culture, l'isolement, la caractérisation des entérovirus et la communication des résultats à ces sites ont été pris en charge par le National Institute of Virology Mumbai Unit, conformément au protocole du Projet national de surveillance de la poliomyélite de l'OMS. Nous avons consacré la première phase de l'étude, qui s'est déroulée entre janvier 2020 et décembre 2021, à la mise en œuvre du protocole au sein de différents établissements médicaux sur sept sites participants, afin de déterminer le nombre d'infections au poliovirus chez les patients souffrant d'un syndrome d'immunodéficience primaire en Inde. Nous avons ensuite, durant la deuxième phase comprise entre janvier 2022 et décembre 2023, élargi l'étude en incluant 14 établissements supplémentaires à travers le pays. Nous sommes convaincus que ce protocole d'étude aidera d'autres pays à instaurer une surveillance du poliovirus dérivé d'une souche vaccinale et lié à une immunodéficience, qui leur servira à identifier et suivre les patients responsables d'une excrétion prolongée du poliovirus d'origine vaccinale. L'intégration, au sein du réseau existant dédié au poliovirus, d'une surveillance de ce type couplée à une surveillance de la paralysie flasque aiguë améliorera le dépistage systématique des patients atteints d'un syndrome d'immunodéficience primaire à l'avenir.

Las personas con inmunodeficiencias primarias infectadas por los poliovirus de origen vacunal pueden seguir excretando poliovirus durante meses sin que la vigilancia de la parálisis flácida aguda los detecte. Por lo tanto, estos pacientes suponen un riesgo de iniciar brotes de poliovirus que pongan en peligro los esfuerzos hacia la erradicación mundial de la poliomielitis. Para identificar a estas personas, diseñamos un protocolo de estudio para el establecimiento de una red de vigilancia de poliovirus de origen vacunal relacionados con inmunodeficiencias en la India. En el primer paso identificamos centros reconocidos en la India que pudieran diagnosticar e inscribir en el estudio a pacientes con trastorno de inmunodeficiencia primaria. La recogida de muestras de heces de los centros de estudio, el cultivo, el aislamiento, la caracterización de los enterovirus y la notificación a los centros de estudio se llevaron a cabo en el Instituto Nacional de Virología, Unidad de Mumbai, según el protocolo del Proyecto Nacional de Vigilancia de la Poliomielitis de la OMS. En la primera fase del estudio, de enero de 2020 a diciembre de 2021, aplicamos el protocolo en siete centros de estudio de diferentes institutos médicos para determinar la proporción de infecciones por poliovirus en pacientes con trastorno de inmunodeficiencia primaria de la India. A continuación, ampliamos el estudio con la inclusión de otros 14 institutos médicos de todo el país en la segunda fase, de enero de 2022 a diciembre de 2023. Creemos que este protocolo de estudio ayudará a otros países a iniciar la vigilancia de poliovirus de origen vacunal relacionados con la inmunodeficiencia para identificar y hacer un seguimiento de los pacientes que son excretores a largo plazo de poliovirus de origen vacunal. La integración de la vigilancia del poliovirus asociado a la inmunodeficiencia con la vigilancia de la parálisis flácida aguda de la red de poliovirus existente mejorará el cribado continuo de pacientes con trastorno por inmunodeficiencia primaria en el futuro.

Genetic, Immunological, and Clinical Features of 32 Patients with Autosomal Recessive STAT1 Deficiency.

Autosomal recessive (AR) STAT1 deficiency is a severe inborn error of immunity disrupting cellular responses to type I, II, and III IFNs, and IL-27, and conferring a predisposition to both viral and mycobacterial infections. We report the genetic, immunological, and clinical features of an international cohort of 32 patients from 20 kindreds: 24 patients with complete deficiency, and 8 patients with partial deficiency. Twenty-four patients suffered from mycobacterial disease (bacillus Calmette-Guérin = 13, environmental mycobacteria = 10, or both in 1 patient). Fifty-four severe viral episodes occurred in sixteen patients, mainly caused by Herpesviridae viruses. Attenuated live measles, mumps, and rubella and/or varicella zoster virus vaccines triggered severe reactions in the five patients with complete deficiency who were vaccinated. Seven patients developed features of hemophagocytic syndrome. Twenty-one patients died, and death was almost twice as likely in patients with complete STAT1 deficiency than in those with partial STAT1 deficiency. All but one of the eight survivors with AR complete deficiency underwent hematopoietic stem cell transplantation. Overall survival after hematopoietic stem cell transplantation was 64%. A diagnosis of AR STAT1 deficiency should be considered in children with mycobacterial and/or viral infectious diseases. It is important to distinguish between complete and partial forms of AR STAT1 deficiency, as their clinical outcome and management differ significantly.

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.

SLGT2 Inhibitor Rescues Myelopoiesis in G6PC3 Deficiency.

The energy metabolism of myeloid cells depends primarily on glycolysis. 1,5-Anhydroglucitol (1,5AG), a natural monosaccharide, is erroneously phosphorylated by glucose-phosphorylating enzymes to produce 1,5-anhydroglucitol-6-phosphate (1,5AG6P), a powerful inhibitor of hexokinases. The endoplasmic reticulum transporter (SLC37A4/G6PT) and the phosphatase G6PC3 cooperate to dephosphorylate 1,5AG6P. Failure to eliminate 1,5AG6P is the mechanism of neutrophil dysfunction and death in G6PC3-deficient mice. Sodium glucose cotransporter 2 (SLGT2) inhibitor reduces 1,5AG level in the blood and restores the neutrophil count in G6PC3-deficient mice. In the investigator-initiated study, a 30-year-old G6PC3-deficient woman with recurrent infections, distressing gastrointestinal symptoms, and multi-lineage cytopenia was treated with an SLGT2-inhibitor. A significant increase in all the hematopoietic cell lineages and substantial improvement in the quality of life was observed.

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.

X-Linked Hyper IgM Syndrome Presenting with Recurrent Tuberculosis-a Case Report.

The hyper IgM syndromes are a group of rare primary immunodeficiency disorders. Currently 6 classes of HIGM are described. X-linked HIGM is also called the type 1 HIGM is the commonest variant in which children present in early infancy with features of combined immunodeficiency. Tuberculosis is a very rare presentation as a presenting symptom in HIGM. Here, we describe a child with XHIGM with recurrent tuberculosis.

Human BCL10 Deficiency due to Homozygosity for a Rare Allele.

In 2014, a child with broad combined immunodeficiency (CID) who was homozygous for a private BCL10 allele was reported to have complete inherited human BCL10 deficiency. In the present study, we report a new BCL10 mutation in another child with CID who was homozygous for a BCL10 variant (R88X), previously reported as a rare allele in heterozygosis (minor allele frequency, 0.000003986). The mutant allele was a loss-of-expression and loss-of-function allele. As with the previously reported patient, this patient had complete BCL10 deficiency. The clinical phenotype shared features, such as respiratory infections, but differed from that of the previous patient that he did not develop significant gastroenteritis episodes or chronic colitis. Cellular and immunological phenotypes were similar to those of the previous patient. TLR4, TLR2/6, and Dectin-1 responses were found to depend on BCL10 in fibroblasts, and final maturation of T cell and B cell maturation into memory cells was affected. Autosomal-recessive BCL10 deficiency should therefore be considered in children with CID.

Approach to Molecular Diagnosis of Chronic Granulomatous Disease (CGD): an Experience from a Large Cohort of 90 Indian Patients.

BACKGROUND: Chronic granulomatous disease (CGD) is characterized by mutation in any one of the five genes coding NADPH oxidase components that leads to functional abnormality preventing the killing of phagocytosed microbes by affecting the progression of a respiratory burst. CGD patients have an increased susceptibility to infections by opportunistic and pathogenic organisms. Though initial diagnosis of CGD using a nitroblue tetrazolium (NBT) test or dihydrorhodamine (DHR) test is relatively easy, molecular diagnosis is challenging due to involvement of multiple genes, presence of pseudogenes, large deletions, and GC-rich regions, among other factors. The strategies for molecular diagnosis vary depending on the affected gene and the mutation pattern prevalent in the target population. There is a paucity of molecular data related to CGD for Indian population. METHOD: This report includes data for a large cohort of CGD patients (n = 90) from India, describing the diagnostic approach, mutation spectrum, and novel mutations identified. We have used mosaicism in mothers and the expression pattern of different NADPH components by flow cytometry as a screening tool to identify the underlying affected gene. The techniques like Sanger sequencing, next-generation sequencing (NGS), and Genescan analysis were used for further molecular analysis. RESULT: Of the total molecularly characterized patients (n = 90), 56% of the patients had a mutation in the NCF1 gene, 30% had mutation in the CYBB gene, and 7% each had mutation in the CYBA and NCF2 genes. Among the patients with NCF1 gene mutation, 82% of the patients had 2-bp deletion (DelGT) mutations in the NCF1 gene. In our cohort, 41 different mutations including 9 novel mutations in the CYBB gene and 2 novel mutations each in the NCF2, CYBA, and NCF1 genes were identified. CONCLUSION: Substantial number of the patients lack NCF1 gene on both the alleles. This is often missed by advanced molecular techniques like Sanger sequencing and NGS due to the presence of pseudogenes and requires a simple Genescan method for confirmation. Thus, the diagnostic approach may depend on the prevalence of affected genes in respective population. This study identifies potential gene targets with the help of flow cytometric analysis of NADPH oxidase components to design an algorithm for diagnosis of CGD in India. In Indian population, the Genescan method should be preferred as the primary molecular test to rule out NCF1 gene mutations prior to Sanger sequencing and NGS.

Poliovirus Excretion in Children with Primary Immunodeficiency Disorders, India.

Prolonged excretion of poliovirus can occur in immunodeficient patients who receive oral polio vaccine, which may lead to propagation of highly divergent vaccine-derived polioviruses (VDPVs), posing a concern for global polio eradication. This study aimed to estimate the proportion of primary immunodeficient children with enterovirus infection and to identify the long-term polio/nonpolio enterovirus excreters in a tertiary care unit in Mumbai, India. During September 2014-April 2017, 151 patients received diagnoses of primary immunodeficiency (PID). We isolated 8 enteroviruses (3 polioviruses and 5 nonpolio enteroviruses) in cell culture of 105 fecal samples collected from 42 patients. Only 1 patient with severe combined immunodeficiency was identified as a long-term VDPV3 excreter (for 2 years after identification of infection). Our results show that the risk of enterovirus excretion among children in India with PID is low; however, systematic screening is necessary to identify long-term poliovirus excreters until the use of oral polio vaccine is stopped.

Clinical, Immunological, and Molecular Findings of Patients with p47phox Defect Chronic Granulomatous Disease (CGD) in Indian Families.

Chronic granulomatous disease (CGD) is a group of inherited disorder of phagocytes, resulting from mutations in the components of the NADPH oxidase complex. Reduced or absent oxygen radical synthesis seen in these patients leads to impaired killing of intracellular bacteria and fungi. CGD clinically presents with recurrent and life-threatening infections as well as granulomatous inflammatory responses. p47phox encoded by the NCF1 gene is the most common autosomal recessive form of CGD which is often clinically milder. Here, we are presenting the data on clinical and immunological findings in 21 Indian patients with Del GT mutation in the NCF1 gene. Diagnosis of these patients was based on detailed clinical evaluation, measurement of respiratory burst activity by nitro blue tetrazolium and dihydrorhodamine-1,2,3 assay, expression of p47phox by flow cytometry, and molecular confirmation by GeneScan method. Seventeen male and four female patients with median age of onset of 1 year ranging from 1.5 months to 6 years were included in the study. Sixty-two percent (13 out of 21) of patients belonged to a consanguineous marriage with only one family having a history of a previous sibling death. Significant variability in clinical presentation was observed in spite of identical genetic defect ranging from asymptomatic to very severe presentation leading to early death or requiring transplantation. However, none of these patients showed difference in immunological parameters to account for this variability. Thus, this study highlights the phenotypic heterogeneity seen in these patients with Del GT mutation in the NCF1 gene and its implication in management of these patients.

Pasteurization of bone for tumour eradication prior to reimplantation - an in vitro & pre-clinical efficacy study.

BACKGROUND & OBJECTIVES: In current era of limb-salvage therapy, pasteurization of bone sarcomas is receiving growing attention as a potential extracorporeal treatment and cost-effective alternative to allografts and radiation before surgical reimplantation. Detailed in vitro and in vivo pre-clinical study to evaluate efficacy of pasteurization to eradicate malignant cells has not been reported yet. The present study was carried out to assess the efficacy of pasteurization to kill tumour cells both in vitro and in vivo. METHODS: Surgically resected specimens of osteosarcomas (n=4) were cut into equal halves and one section was pasteurized by heating at 60°C to 65°C for 40 min. Paired samples before and after pasteurization were studied in vitro for DNA ploidy, evaluation of histological change and elimination of mitotic activity. These tissues were transplanted in immune-deficient NOD-SCID mice to evaluate effect on tumour-generating ability, presence of human nuclei, osteopontin and cytokine/chemokines released in tumour-transplanted mice. RESULTS: Non-pasteurized tumour samples had viable tumour cells which exhibited significant growth in culture, increased proliferative ability and clonogenic potential while respective pasteurized tumour tissues did not grow in culture and did not exhibit clonogenicity. Flow cytometry revealed that propidium iodide positive dead cells increased significantly (P< 0.01) post pasteurization. Seven of 12 non-pasteurized tumour transplanted mice demonstrated tumour-forming ability as against 0 of 12 in pasteurized tumour transplanted mice. Solid tumour xenografts exhibited strong expression of anti-human nuclei and osteopontin by immunohistochemistry as well as secretary human interluekin-6 (IL-6) while pasteurized mice failed to express these markers. INTERPRETATION & CONCLUSIONS: This study has provided a basis to establish pasteurization as being efficacious in ensuring tumour eradication from resected bone tumour specimens. Pasteurized tumour bearing bone can thus safely be used to reconstruct large defects after tumour resection.