A case of T-cell-Epstein-Barr virus-haemophagocytic lymphohistiocytosis and sustained remission following ruxolitinib therapy.

Objectives: Epstein-Barr virus (EBV) is a common cause of secondary haemophagocytic lymphohistiocytosis (HLH). While B cells are reservoirs for EBV, infection within T cells and NK cells in this disease can be difficult to treat. Methods: A 19-year-old female presented with a 6-week history of coryzal symptoms on a background of Crohn's disease. On examination, she was febrile and tachycardic with mild tonsillar enlargement and splenomegaly. New trilineage cytopenias and elevation in liver enzymes were detected, with acute EBV subsequently confirmed on whole blood PCR. A diagnosis of EBV-associated HLH was supported further with elevated serum ferritin, triglycerides and soluble CD25, low fibrinogen and the presence of haemophagocytosis in the bone marrow. Results: Corticosteroids, IVIG and rituximab were given, and anakinra was subsequently added due to ongoing fevers. EBV infection was then demonstrated within CD8+ T cells on EBER Flow-FISH assay. Ruxolitinib was commenced and her fevers abated on day 5, with improvement in other HLH parameters. She was discharged after a 39-day hospital admission. To date, she has remained in remission of HLH, despite developing COVID-19 infection during the convalescence phase of HLH. Conclusion: EBV viraemia requires adequate treatment to control EBV-associated HLH as rituximab may be insufficient, and corticosteroid resistance can result in continued EBV infection in CD8+ T cells. This entity is known as T-cell-EBV-HLH. Ruxolitinib is a novel treatment strategy in this specific context and has several advantages, including inhibition of corticosteroid resistance to promote apoptosis of EBV-infected T cells.

CTLA4 protects against maladaptive cytotoxicity during the differentiation of effector and follicular CD4+ T cells.

As chronic antigenic stimulation from infection and autoimmunity is a feature of primary antibody deficiency (PAD), analysis of affected patients could yield insights into T-cell differentiation and explain how environmental exposures modify clinical phenotypes conferred by single-gene defects. CD57 marks dysfunctional T cells that have differentiated after antigenic stimulation. Indeed, while circulating CD57+ CD4+ T cells are normally rare, we found that they are increased in patients with PAD and markedly increased with CTLA4 haploinsufficiency or blockade. We performed single-cell RNA-seq analysis of matched CD57+ CD4+ T cells from blood and tonsil samples. Circulating CD57+ CD4+ T cells (CD4cyt) exhibited a cytotoxic transcriptome similar to that of CD8+ effector cells, could kill B cells, and inhibited B-cell responses. CTLA4 restrained the formation of CD4cyt. While CD57 also marked an abundant subset of follicular helper T cells, which is consistent with their antigen-driven differentiation, this subset had a pre-exhaustion transcriptomic signature marked by TCF7, TOX, and ID3 expression and constitutive expression of CTLA4 and did not become cytotoxic even after CTLA4 inhibition. Thus, CD57+ CD4+ T-cell cytotoxicity and exhaustion phenotypes are compartmentalised between blood and germinal centers. CTLA4 is a key modifier of CD4+ T-cell cytotoxicity, and the pathological CD4cyt phenotype is accentuated by infection.

Pneumococcal IgG Antibody Responses to 23vPPV in Healthy Controls Using an Automated ELISA.

Measurement of pre- and post-pneumococcal antibody levels after immunization with the 23-valent capsular polysaccharide pneumococcal vaccine (23vPPV) is indicative of a T-independent antibody response. The World Health Organisation ELISA is considered gold standard yet is labor-intensive and technically difficult to perform. Interpretation criteria defining an adequate response to 23vPPV remain controversial. The diagnostic Immunology Laboratory at The Royal Children's Hospital, Melbourne (RCH), performs an in-house multi-serotype automated ELISA. The primary objective of this study was to verify RCH interpretation criteria for the laboratory's automated ELISA. Forty pneumococcal conjugate vaccine (PCV)-naïve healthy adults aged 18 to 25 years and 22 PCV-primed healthy children aged 2 to 5 years were immunized with 23vPPV. A serum sample was collected immediately prior and 28 to 42 (± 7) days post immunization. Samples were analyzed on the Tecan Freedom Evo 200 ELISA with adequate response defined as post-immunization antibody level of 1.3 µg/mL or fourfold rise from baseline in ≥ 10/15 serotypes in adult participants and ≥ 4/8 serotypes in pediatric participants. Thirty-nine (97.5%) adults and 22 (100%) children achieved an adequate response to 23vPPV. In PCV-naïve adults, serotypes contained within the conjugate vaccines were less immunogenic, with 12 (30%) adults not achieving an adequate antibody response when only PCV serotypes were used for interpretation. Our diagnostic laboratory has verified the interpretation criteria used for an automated multi-serotype pneumococcal ELISA method. Clinical Trial Registration: ANZCTR registration number ACTRN12618000822280.

Rubella vaccine-induced granulomas are a novel phenotype with incomplete penetrance of genetic defects in cytotoxicity.

BACKGROUND: Rubella virus-induced granulomas have been described in patients with various inborn errors of immunity. Most defects impair T-cell immunity, suggesting a critical role of T cells in rubella elimination. However, the molecular mechanism of virus control remains elusive. OBJECTIVE: This study sought to understand the defective effector mechanism allowing rubella vaccine virus persistence in granulomas. METHODS: Starting from an index case with Griscelli syndrome type 2 and rubella skin granulomas, this study combined an international survey with a literature search to identify patients with cytotoxicity defects and granuloma. The investigators performed rubella virus immunohistochemistry and PCR and T-cell migration assays. RESULTS: This study identified 21 patients with various genetically confirmed cytotoxicity defects, who presented with skin and visceral granulomas. Rubella virus was demonstrated in all 12 accessible biopsies. Granuloma onset was typically before 2 years of age and lesions persisted from months to years. Granulomas were particularly frequent in MUNC13-4 and RAB27A deficiency, where 50% of patients at risk were affected. Although these proteins have also been implicated in lymphocyte migration, 3-dimensional migration assays revealed no evidence of impaired migration of patient T cells. Notably, patients showed no evidence of reduced control of concomitantly given measles, mumps, or varicella live-attenuated vaccine or severe infections with other viruses. CONCLUSIONS: This study identified lymphocyte cytotoxicity as a key effector mechanism for control of rubella vaccine virus, without evidence for its need in control of live measles, mumps, or varicella vaccines. Rubella vaccine-induced granulomas are a novel phenotype with incomplete penetrance of genetic disorders of cytotoxicity.

Reduced-Intensity/Reduced-Toxicity Conditioning Approaches Are Tolerated in XIAP Deficiency but Patients Fare Poorly with Acute GVHD.

X-linked inhibitor of apoptosis (XIAP) deficiency is an inherited primary immunodeficiency characterized by chronic inflammasome overactivity and associated with hemophagocytic lymphohistiocytosis (HLH) and inflammatory bowel disease (IBD). Allogeneic hematopoietic cell transplantation (HCT) with fully myeloablative conditioning may be curative but has been associated with poor outcomes. Reports of reduced-intensity conditioning (RIC) and reduced-toxicity conditioning (RTC) regimens suggest these approaches are well tolerated, but outcomes are not well established. Retrospective data were collected from an international cohort of 40 patients with XIAP deficiency who underwent HCT with RIC or RTC. Thirty-three (83%) patients had a history of HLH, and thirteen (33%) patients had IBD. Median age at HCT was 6.5 years. Grafts were from HLA-matched (n = 30, 75%) and HLA-mismatched (n = 10, 25%) donors. There were no cases of primary graft failure. Two (5%) patients experienced secondary graft failure, and three (8%) patients ultimately received a second HCT. Nine (23%) patients developed grade II-IV acute GVHD, and 3 (8%) developed extensive chronic GVHD. The estimated 2-year overall and event-free survival rates were 74% (CI 55-86%) and 64% (CI 46-77%), respectively. Recipient and donor HLA mismatch and grade II-IV acute GVHD were negatively associated with survival on multivariate analysis with hazard ratios of 5.8 (CI 1.5-23.3, p = 0.01) and 8.2 (CI 2.1-32.7, p < 0.01), respectively. These data suggest that XIAP patients tolerate RIC and RTC with survival rates similar to HCT of other genetic HLH disorders. Every effort should be made to prevent acute GVHD in XIAP-deficient patients who undergo allogeneic HCT.

Impact of antibiotic allergy labels on patient outcomes in a tertiary paediatric hospital.

AIMS: Antibiotic allergies are reported in 5-15% of children. This study aimed to evaluate the impact of common ß-lactam antibiotic allergy labels (AALs) on hospital treatment, focusing on length of stay and appropriateness of antibiotic prescribing. METHODS: This was a retrospective cohort study over 21 months at the Royal Children's Hospital Melbourne, Australia. A subset of children with the most common ß-lactam allergies, and who required admission for intravenous antibiotics over a 12-month period, was analysed for appropriateness of prescribing. Non-allergic patients were matched to evaluate associations between AALs and hospital treatment. RESULTS: There were 98 912 children admitted over the study period, of whom 938 (1%) had at least one AAL on first admission. Of all encounters, 5145 (2.5%) were for children with AALs. The most common AALs were to amoxicillin and amoxicillin-clavulanic acid combinations (40.8%), cefalexin (14.4%) and trimethoprim-sulfamethoxazole (9.7%). For the subset, there were 66 admissions for children who required intravenous antibiotics. Documentation was adequate for 27% of AALs. Inappropriate prescribing occurred in almost half (47%). Hospital stay was longer for children with AALs (median 4.7 days; IQR 2.3-9.2) compared to non-allergic controls (median 3.9 days; IQR 1.9-6.8; P = .02). Children with AALs were more likely to receive restricted antibiotics (aOR 3.03; 95% CI, 1.45-6.30; P = .003). CONCLUSION: This is the first study to demonstrate high rates of inappropriate prescribing in children with AALs. Children with AALs were significantly more likely to receive restricted antibiotics and had a longer length of stay compared with non-allergic controls.

Extended clinical and immunological phenotype and transplant outcome in CD27 and CD70 deficiency.

Biallelic mutations in the genes encoding CD27 or its ligand CD70 underlie inborn errors of immunity (IEIs) characterized predominantly by Epstein-Barr virus (EBV)-associated immune dysregulation, such as chronic viremia, severe infectious mononucleosis, hemophagocytic lymphohistiocytosis (HLH), lymphoproliferation, and malignancy. A comprehensive understanding of the natural history, immune characteristics, and transplant outcomes has remained elusive. Here, in a multi-institutional global collaboration, we collected the clinical information of 49 patients from 29 families (CD27, n = 33; CD70, n = 16), including 24 previously unreported individuals and identified a total of 16 distinct mutations in CD27, and 8 in CD70, respectively. The majority of patients (90%) were EBV+ at diagnosis, but only ∼30% presented with infectious mononucleosis. Lymphoproliferation and lymphoma were the main clinical manifestations (70% and 43%, respectively), and 9 of the CD27-deficient patients developed HLH. Twenty-one patients (43%) developed autoinflammatory features including uveitis, arthritis, and periodic fever. Detailed immunological characterization revealed aberrant generation of memory B and T cells, including a paucity of EBV-specific T cells, and impaired effector function of CD8+ T cells, thereby providing mechanistic insight into cellular defects underpinning the clinical features of disrupted CD27/CD70 signaling. Nineteen patients underwent allogeneic hematopoietic stem cell transplantation (HSCT) prior to adulthood predominantly because of lymphoma, with 95% survival without disease recurrence. Our data highlight the marked predisposition to lymphoma of both CD27- and CD70-deficient patients. The excellent outcome after HSCT supports the timely implementation of this treatment modality particularly in patients presenting with malignant transformation to lymphoma.

Activated PI3Kδ breaches multiple B cell tolerance checkpoints and causes autoantibody production.

Antibody-mediated autoimmune diseases are a major health burden. However, our understanding of how self-reactive B cells escape self-tolerance checkpoints to secrete pathogenic autoantibodies remains incomplete. Here, we demonstrate that patients with monogenic immune dysregulation caused by gain-of-function mutations in PIK3CD, encoding the p110δ catalytic subunit of phosphoinositide 3-kinase (PI3K), have highly penetrant secretion of autoreactive IgM antibodies. In mice with the corresponding heterozygous Pik3cd activating mutation, self-reactive B cells exhibit a cell-autonomous subversion of their response to self-antigen: instead of becoming tolerized and repressed from secreting autoantibody, Pik3cd gain-of-function B cells are activated by self-antigen to form plasmablasts that secrete high titers of germline-encoded IgM autoantibody and hypermutating germinal center B cells. However, within the germinal center, peripheral tolerance was still enforced, and there was selection against B cells with high affinity for self-antigen. These data show that the strength of PI3K signaling is a key regulator of pregerminal center B cell self-tolerance and thus represents a druggable pathway to treat antibody-mediated autoimmunity.

Non-ß-Lactam Antibiotic Hypersensitivity Reactions.

OBJECTIVES: Antibiotics are among the most common prescriptions in children, and non-ß-lactam antibiotics (NBLAs) account for almost half of those prescribed in Australian pediatric hospitals. Despite this, data on NBLA hypersensitivity in children are limited. This study describes reported hypersensitivity reactions to NBLAs in children and the results of allergy evaluation. METHODS: Children with a suspected NBLA allergy who had skin testing and/or an intravenous or oral challenge test (OCT) between May 2011 and June 2018 were included. Patients were excluded if they were >18 years old or did not complete the allergy evaluation for any reason other than allergic reaction. RESULTS: Over the 7-year study period, 141 children had 150 allergy evaluations of 15 different NBLAs. The median time from the initial reported reaction to allergy evaluation was 1.9 (range 0.1-14.9) years. Overall, 27 of the 150 (18.0%) challenge tests to NBLAs had positive results, with the rate of positive OCT results being highest for trimethoprim-sulfamethoxazole (15 of 46; 32.6%) and macrolides (8 of 77; 10.4%). Although 4 children reported initial anaphylactic reactions, no patients had severe symptoms on rechallenge or required adrenaline. Of the challenges that had positive results, the majority of children (23 of 27; 85.2%) had symptoms on repeat challenge similar to those that were initially reported. CONCLUSIONS: Overall, 8 of 10 children with NBLA allergy could be delabeled. On average, patients waited 1.9 years to be rechallenged. Timely access to allergy evaluation to delabel these patients is needed to preserve first-line antibiotics.

Use of TCR α+ß+/CD19+-Depleted Haploidentical Hematopoietic Stem Cell Transplant Is a Viable Option in Patients With Primary Immune Deficiency Without Matched Sibling Donor.

Allogeneic hematopoietic stem cell transplantation (HSCT) is curative for many patients with primary immune deficiency (PID). Haploidentical donors have historically been associated with higher rates of graft-versus-host disease (GvHD) and graft failure. Use of T cell receptor (TCR) α+ß+/CD19+-depleted grafts has resulted in improved haploidentical HSCT outcomes. We sought to evaluate outcomes of TCR α+ß+/CD19+-depleted haploidentical HSCT in pediatric patients with PID at a single center in Australia. Specifically, we evaluated immune reconstitution, looking at time to T cell and B cell reconstitution, and B cell function post-HSCT. Eleven patients with a mean age of 7.92 years (range 0.33-17.17 years) were included. The median time to B cell recovery was 93 days (range 41-205 days), and the median time to cessation of immunoglobulin replacement was 281.5 days (range 41-205 days). All patients who had ceased immunoglobulin replacement had an adequate response to pneumococcal conjugate (Prevenar 13) vaccine. The median time to CD4+ recovery was 132 days (range 30-296 days), and naive T cells were present in all surviving patients by 4 months post-HSCT. Eight of 11 patients are surviving, with six patients having whole blood chimerism greater than 95%, one patient with whole blood chimerism of 82.8%, and another with 76.0%. All of these patients clinically had no evidence of underlying immunodeficiency. Likelihood of overall survival at 2 years post-HSCT was 81.8%. Cumulative incidence of acute GvHD was 27.3%. Cumulative incidence of CMV viremia was 63.6%. All patients previously exposed to CMV had reactivation post-HSCT, but were controlled with pre-emptive CMV treatment. Assuming most children with PID have a haploidentical donor available, use of this technique is likely to result in good outcomes for patients who do not have a suitable matched sibling or matched unrelated donor.

Hematopoietic stem cell transplant effectively rescues lymphocyte differentiation and function in DOCK8-deficient patients.

Bi-allelic inactivating mutations in DOCK8 cause a combined immunodeficiency characterised by severe pathogen infections, eczema, allergies, malignancy and impaired humoral responses. These clinical features result from functional defects in most lymphocyte lineages. Thus, DOCK8 plays a key role in immune cell function. Hematopoietic stem cell transplantation (HSCT) is curative for DOCK8 deficiency. While previous reports have described clinical outcomes for DOCK8 deficiency following HSCT, the effect on lymphocyte reconstitution and function has not been investigated. Our study determined whether defects in lymphocyte differentiation and function in DOCK8-deficient patients were restored following HSCT. DOCK8-deficient T and B lymphocytes exhibited aberrant activation and effector function in vivo and in vitro. Frequencies of αß T and MAIT cells were reduced while γδT cells were increased in DOCK8-deficient patients. HSCT improved, abnormal lymphocyte function in DOCK8-deficient patients. Elevated total and allergen-specific IgE in DOCK8-deficient patients decreased over time following HSCT. Our results document the extensive catalogue of cellular defects in DOCK8-deficient patients, and the efficacy of HSCT to correct these defects, concurrent with improvements in clinical phenotypes. Overall, our findings provide mechanisms at a functional cellular level for improvements in clinical features of DOCK8 deficiency post-HSCT, identify biomarkers that correlate with improved clinical outcomes, and inform the general dynamics of immune reconstitution in patients with monogenic immune disorders following HSCT.

Activating mutations in PIK3CD disrupt the differentiation and function of human and murine CD4+ T cells.

BACKGROUND: Gain-of-function (GOF) mutations in PIK3CD cause a primary immunodeficiency characterized by recurrent respiratory tract infections, susceptibility to herpesvirus infections, and impaired antibody responses. Previous work revealed defects in CD8+ T and B cells that contribute to this clinical phenotype, but less is understood about the role of CD4+ T cells in disease pathogenesis. OBJECTIVE: We sought to dissect the effects of increased phosphoinositide 3-kinase (PI3K) signaling on CD4+ T-cell function. METHODS: We performed detailed ex vivo, in vivo, and in vitro phenotypic and functional analyses of patients' CD4+ T cells and a novel murine disease model caused by overactive PI3K signaling. RESULTS: PI3K overactivation caused substantial increases in numbers of memory and follicular helper T (TFH) cells and dramatic changes in cytokine production in both patients and mice. Furthermore, PIK3CD GOF human TFH cells had dysregulated phenotype and function characterized by increased programmed cell death protein 1, CXCR3, and IFN-γ expression, the phenotype of a TFH cell subset with impaired B-helper function. This was confirmed in vivo in which Pik3cd GOF CD4+ T cells also acquired an aberrant TFH phenotype and provided poor help to support germinal center reactions and humoral immune responses by antigen-specific wild-type B cells. The increase in numbers of both memory and TFH cells was largely CD4+ T-cell extrinsic, whereas changes in cytokine production and TFH cell function were cell intrinsic. CONCLUSION: Our studies reveal that CD4+ T cells with overactive PI3K have aberrant activation and differentiation, thereby providing mechanistic insight into dysfunctional antibody responses in patients with PIK3CD GOF mutations.

Vaccine Allergy? Skin Testing and Challenge at a Tertiary Pediatric Hospital in Melbourne, Australia.

BACKGROUND: The rate of true vaccine allergy is unknown. Children with potential IgE-mediated adverse events following immunization (AEFI) should undergo allergy investigation that may include skin testing or challenge. Previous protocols tend to be highly conservative and often suggest invasive testing for all, a practice not evidence based, technically difficult, and unpleasant in children. It has more recently been suggested that skin testing may be restricted to those with allergic-like events within the first hour and those with a history of anaphylaxis. OBJECTIVE: We aimed to describe the outcome of vaccine skin testing and challenge in children referred to a tertiary pediatric hospital with a potential IgE-mediated AEFI. The secondary aim was to identify any significant risk factors for vaccine allergy. METHODS: A retrospective review of all children (<18 years) who underwent vaccine skin testing (skin prick testing or intradermal testing [IDT]) or challenge over a 5-year period (May 1, 2011, to April 30, 2016) at the Royal Children's Hospital Melbourne is presented. RESULTS: There were 95 admissions in 73 children. Eight percent (6 of 73) of children had confirmed vaccine allergy (positive skin testing or challenge to the index vaccination). Two had positive IDT to a suspect vaccine but challenge negative to an alternative brand vaccine. Two had negative IDT but subsequent positive challenge and two had immediate urticaria on challenge without prior skin testing. All children in the positive group either had index reaction within 15 minutes of vaccination or had history consistent with anaphylaxis. CONCLUSIONS: The vast majority of children (92%) presenting with a potential IgE-mediated AEFI are able to tolerate challenge to a suspect vaccine without reaction. We present our investigation protocol recommending skin testing in all children with anaphylaxis and challenge with a suspect vaccine if negative testing or previous nonanaphylactic potential IgE-mediated AEFI.

Activating PIK3CD mutations impair human cytotoxic lymphocyte differentiation and function and EBV immunity.

BACKGROUND: Germline gain-of function (GOF) mutations in PIK3CD, encoding the catalytic p110δ subunit of phosphoinositide 3-kinase (PI3K), result in hyperactivation of the PI3K-AKT-mechanistic target of rapamycin pathway and underlie a novel inborn error of immunity. Affected subjects exhibit perturbed humoral and cellular immunity, manifesting as recurrent infections, autoimmunity, hepatosplenomegaly, uncontrolled EBV and/or cytomegalovirus infection, and increased incidence of B-cell lymphoproliferation, lymphoma, or both. Mechanisms underlying disease pathogenesis remain unknown. OBJECTIVE: Understanding the cellular and molecular mechanisms underpinning inefficient surveillance of EBV-infected B cells is required to understand disease in patients with PIK3CD GOF mutations, identify key molecules required for cell-mediated immunity against EBV, and develop immunotherapeutic interventions for the treatment of this and other EBV-opathies. METHODS: We studied the consequences of PIK3CD GOF mutations on the generation, differentiation, and function of CD8+ T cells and natural killer (NK) cells, which are implicated in host defense against infection with herpesviruses, including EBV. RESULTS: PIK3CD GOF total and EBV-specific CD8+ T cells were skewed toward an effector phenotype, with exaggerated expression of markers associated with premature immunosenescence/exhaustion and increased susceptibility to reactivation-induced cell death. These findings were recapitulated in a novel mouse model of PI3K GOF mutations. NK cells in patients with PIK3CD GOF mutations also exhibited perturbed expression of differentiation-associated molecules. Both CD8+ T and NK cells had reduced capacity to kill EBV-infected B cells. PIK3CD GOF B cells had increased expression of CD48, programmed death ligand 1/2, and CD70. CONCLUSIONS: PIK3CD GOF mutations aberrantly induce exhaustion, senescence, or both and impair cytotoxicity of CD8+ T and NK cells. These defects might contribute to clinical features of affected subjects, such as impaired immunity to herpesviruses and tumor surveillance.

Germline-activating mutations in PIK3CD compromise B cell development and function.

Gain-of-function (GOF) mutations in PIK3CD, encoding the p110δ subunit of phosphatidylinositide 3-kinase (PI3K), cause a primary immunodeficiency. Affected individuals display impaired humoral immune responses following infection or immunization. To establish mechanisms underlying these immune defects, we studied a large cohort of patients with PIK3CD GOF mutations and established a novel mouse model using CRISPR/Cas9-mediated gene editing to introduce a common pathogenic mutation in Pik3cd In both species, hyperactive PI3K severely affected B cell development and differentiation in the bone marrow and the periphery. Furthermore, PI3K GOF B cells exhibited intrinsic defects in class-switch recombination (CSR) due to impaired induction of activation-induced cytidine deaminase (AID) and failure to acquire a plasmablast gene signature and phenotype. Importantly, defects in CSR, AID expression, and Ig secretion were restored by leniolisib, a specific p110δ inhibitor. Our findings reveal key roles for balanced PI3K signaling in B cell development and long-lived humoral immunity and memory and establish the validity of treating affected individuals with p110δ inhibitors.

Newborn screening for severe combined immunodeficiency: Evaluation of a commercial T-cell receptor excision circle-based method in Victorian dried blood spots.

AIM: Severe combined immunodeficiency (SCID) is the most severe form of primary immunodeficiency and is fatal in infancy if untreated. As early diagnosis is associated with improved outcomes, SCID is an ideal condition to consider for inclusion in a newborn screening (NBS) programme in Australia. In this feasibility study, we evaluated the EnLite Neonatal TREC kit for detection of T-cell receptor excision circles (TRECs) from NBS dried blood spots for the identification of known SCID patients in Victoria. METHODS: TREC copies/µL were measured retrospectively in 14 children diagnosed with SCID or complete DiGeorge syndrome (CDGS) from 2005 to 2015 at the Royal Children's Hospital, Melbourne. In addition, TREC copies/µL were measured for 501 prospective de-identified NBS cards. RESULTS: Of 14 known SCID or CDGS samples, 11 were correctly identified as presumptive positive samples with low or undetectable TREC on duplicate testing. The remaining three samples also had low or undetectable TREC on duplicate testing but were considered invalid due to insufficient ß-actin DNA amplification. Of the 501 prospective NBS samples, none were identified as presumptive positive samples on duplicate testing. CONCLUSIONS: The EnLite Neonatal TREC kit correctly identified known SCID or CDGS patients as presumptive positive samples, and initial cut-offs for TREC and ß-actin in the Victorian NBS population were determined. A larger pilot study is required to confirm these proposed cut-offs and to evaluate the cost and implementation of this screening programme in Victoria, Australia. Overall, this study provides preliminary data to support the introduction of this assay to the NBS programme in Victoria.