Analysis of Clinical, Immunological and Molecular Features of Leukocyte Adhesion Deficiency Type I in Egyptian Children.

PURPOSE: Leukocyte adhesion deficiency (LAD) represents a rare group of inherited inborn errors of immunity (IEI) characterized by bacterial infections, delayed umbilical stump separation, and autoimmunity. This single-center study aimed at describing the clinical, immunological, and molecular characterizations of 34 LAD-I Egyptian pediatric patients. METHODS: Details of 34 patients' personal medical history, clinical and laboratory findings were recorded; Genetic material from 28 patients was studied. Mutational analysis was done by Sanger sequencing. RESULTS: Omphalitis, skin and soft tissue infections with poorly healing ulcers, delayed falling of the umbilical stump, and recurrent or un-resolving pneumonia were the most common presentations, followed by chronic otitis media, enteropathy, periodontitis; and recurrent oral thrush. Persistent leukocytosis and neutrophilia were reported in all patients, as well as CD18 and CD11b deficiency. CD18 expression was < 2% in around 90% of patients. Sixteen different pathological gene variants were detected in 28 patients who underwent ITGß2 gene sequencing, of those, ten were novel and six were previously reported. Three families received a prenatal diagnosis. Patients were on antimicrobials according to culture's results whenever available, and on prophylactic Trimethoprim-Sulfamethoxazole 5 mg/kg once daily, with regular clinical follow up. Hematopoietic stem cell transplantation (HSCT) was offered for 4 patients. However due to severity of the disease and delay in diagnosis, 58% of the patients passed away in the first 2 years of life. CONCLUSION: This study highlights the importance of early diagnosis and distribution of ITGß2 gene mutation in Egyptian children. Further molecular studies, however, remain a challenging necessity for better disease characterization in the region.

Genetic Testing in Egyptian Patients with Inborn Errors of Immunity: a Single-Center Experience.

BACKGROUND: Inborn errors of immunity (IEI) are a group of heterogeneous disorders with geographic and ethnic diversities. Although IEI are common in Egypt, genetic diagnosis is limited due to financial restrictions. This study aims to characterize the genetic spectrum of IEI patients in Egypt and highlights the adaptation of the molecular diagnostic methods to a resource-limited setting. METHODS: Genetic material from 504 patients was studied, and proper diagnosis was achieved in 282 patients from 246 families. Mutational analysis was done by Sanger sequencing, next-generation sequencing (NGS) targeting customized genes panels, and whole-exome sequencing (WES) according to the patients' phenotypes and availability of genetic testing. RESULTS: A total of 194 variants involving 72 different genes were detected with RAG1/2 genes being the most encountered followed by DOCK8, CYBA, LRBA, NCF1, and JAK3. Autosomal recessive (AR) inheritance was detected in 233/282 patients (82.6%), X-linked (XL) recessive inheritance in 32/282 patients (11.3%), and autosomal dominant (AD) inheritance in 18/282 patients (6.4%), reflecting the impact of consanguineous marriages on the prevalence of different modes of inheritance and the distribution of the various IEI disorders. CONCLUSION: The study showed that a combination of Sanger sequencing in selected patients associated with targeted NGS or WES in other patients is an effective diagnostic strategy for IEI diagnosis in countries with limited diagnostic resources. Molecular testing can be used to validate other nonexpensive laboratory techniques that help to reach definitive diagnosis and help in genetic counseling and taking proper therapeutic decisions including stem cell transplantation or gene therapy.

MHC-II Deficiency Among Egyptians: Novel Mutations and Unique Phenotypes.

BACKGROUND: MHC class II deficiency leads to defective CD4+ T-cell function that results from impaired antigen presentation. A genetic disorder in 1 of 4 genes results in this syndrome that is associated with the clinical phenotype of combined immunodeficiency. OBJECTIVE: To describe the clinical, immunological, and molecular characteristics of 10 Egyptian patients from 9 different families having presented with MHC class II deficiency between 2012 and 2017. METHODS: An initial diagnosis based on the combination of clinical features and low HLA-DR expression by flow cytometry was confirmed by genetic analyses. RESULTS: Symptoms included failure to thrive (n = 9), persistent diarrhea (n = 5), and pneumonia (n = 8). Septicemia due to coagulase-negative staphylococci (n = 1) and Candida krusei (n = 1) was diagnosed. Nine patients orally received the live attenuated polio vaccine, of whom 3 developed acute flaccid paralysis thereafter. Nine patients received the BCG vaccine and none developed obvious signs of BCGitis. Four patients carried RFXANK gene mutations, 3 carried RFX5 gene mutations, 1 carried a CIITA gene mutation, and none carried RFXAP gene mutations. Six of the 7 detected mutations were previously unreported mutations: c.431T>C, c.247_250delTCAG, and c.600delG in the RFXANK gene; c.116+1G>A and c.715C>T in the RFX5 gene; and c.929delA in the CIITA gene. CONCLUSIONS: Given that Egypt is a North African country with a high rate of consanguinity, MHC class II deficiency is not rare. However, the molecular defects differ from those reported in nearby countries. Early diagnosis must be based on suspicious clinical signs and laboratory diagnosis because the defect can be missed by T-cell receptor excision circles based on neonatal screening.

Poliovirus excretion following vaccination with live poliovirus vaccine in patients with primary immunodeficiency disorders: clinicians' perspectives in the endgame plan for polio eradication.

OBJECTIVE: Primary immunodeficiency (PID) patients are prone to developing viral infections and should not be vaccinated with live vaccines. In such patients, prolonged excretion and viral divergence may occur and they may subsequently act as reservoirs in the community introducing mutated virus and jeopardizing polio eradication. One hundred and thirty PID cases were included for poliovirus detection in stool with assessment of divergence of detected polioviruses from oral polio vaccine (OPV) virus. Clinical presentations of PID patients with detectable poliovirus in stool specimens are described. RESULTS: Six PID patients (4.5%) had detectable vaccine-derived poliovirus (VDPV) excretion in stool specimens; of these, five patients had severe combined immunodeficiency (two with acute flaccid paralysis, one with meningoencephalitis and two without neurological manifestations), and one patient had X-linked agammaglobulinemia (paralysis developed shortly after diagnosis of immunodeficiency). All six case-patients received trivalent OPV. Five case-patients had type 2 immunodeficiency-related vaccine-derived polioviruses (iVDPV2) excretion; one had concomitant excretion of Sabin like type 3 virus and one was identified as iVDPV1 excretor. Surveillance for poliovirus excretion among PID patients is critical as these patients represent a potential source to reseed polioviruses into populations.

Diagnosis of DOCK8 deficiency using Flow cytometry Biomarkers: an Egyptian Center experience.

In the past few years, several genes were shown to be implicated in various forms of the Hyper Immunoglobulin E syndrome. The present study is the first to describe a cohort of DOCK8 deficiency patients from Egypt. The study included 15 patients with features of combined immunodeficiency (CID) suggestive of DOCK8 deficiency. Flow cytometry was used for evaluation of DOCK8 expression and studying different immunological characteristics of those patients including evaluation of Th17, Tregs, T and B lymphocytes differentiation and the effect of the DOCK8 deficiency on the activation of the STAT3. Diagnosis was confirmed by mutational analysis. Profound defects in Th17 cells and Tregs were observed in all patients with impaired STAT3 phosphorylation, indicating that DOCK8 plays a pivotal role in the STAT3 signaling pathway. These findings together with decrease in memory B cells and defective DOCK8 expression by flow cytometry can confirm the diagnosis.

Genetic Counseling in Primary Immunodeficiency Disorders: An Emerging Experience in Egypt.

BACKGROUND: Primary immunodeficiency disorders (PIDs) are a heterogeneous group of diseases of the immune system leading to life-threatening infections, and, unless urgently treated with immune reconstitution, patients do not usually survive. With the continuing progress in molecular diagnosis, many mutations have been described in more than 300 genes. Genetic counseling has recently been considered an essential part of the management of PIDs. This study presents the experience of genetic counseling services in the largest PID center in Egypt, and reports on our management plan and the impact of prenatal diagnosis (PND) on families. METHODS: Based on the biochemical and molecular diagnosis of index cases, PND was offered for 10 families in 12 subsequent pregnancies. Five different genes were sequenced by Sanger sequencing in fetal samples. RESULTS: Seven fetuses were either normal or were carriers, while five fetuses were affected and human leukocyte antigen typing was performed, seeking a suitably related donor for stem cell transplantation. CONCLUSION: In spite of the genetic heterogeneity behind PIDs, genetic counseling should play a critical role in the management and future decisions of affected families.

CD4(+) CD25(+) cells in type 1 diabetic patients with other autoimmune manifestations.

The existence of multiple autoimmune disorders in diabetics may indicate underlying primary defects of immune regulation. The study aims at estimation of defects of CD4(+) CD25(+high) cells among diabetic children with multiple autoimmune manifestations, and identification of disease characteristics in those children. Twenty-two cases with type 1 diabetes associated with other autoimmune diseases were recruited from the Diabetic Endocrine and Metabolic Pediatric Unit (DEMPU), Cairo University along with twenty-one normal subjects matched for age and sex as a control group. Their anthropometric measurements, diabetic profiles and glycemic control were recorded. Laboratory investigations included complete blood picture, glycosylated hemoglobin, antithyroid antibodies, celiac antibody panel and inflammatory bowel disease markers when indicated. Flow cytometric analysis of T-cell subpopulation was performed using anti-CD3, anti-CD4, anti-CD8, anti-CD25 monoclonal antibodies. Three cases revealed a proportion of CD4(+) CD25(+high) below 0.1% and one case had zero counts. However, this observation did not mount to a significant statistical difference between the case and control groups neither in percentage nor absolute numbers. Significant statistical differences were observed between the case and the control groups regarding their height, weight centiles, as well as hemoglobin percentage, white cell counts and the absolute lymphocytic counts. We concluded that, derangements of CD4(+) CD25(+high) cells may exist among diabetic children with multiple autoimmune manifestations indicating defects of immune controllers.

Pattern of intravenous immunoglobulins (IVIG) use in a pediatric intensive care facility in a resource limited setting.

BACKGROUND: Intravenous Immunoglobulin (IVIG) preparations are scarce biological products used for replacement or immunomodulatory effects. Guidelines have been issued by regulatory health authorities to ensure provision of the products for patients who are in severe need. OBJECTIVES: The study aimed at description of the pattern of IVIG use (label/off label indications), adverse effects observed, reason for choice of IVIG among other modalities and efficacy in a pediatric intensive care setting. METHODS: A retrospective chart review. PATIENTS: The study included 45 cases admitted from 2008 through 2011 in a Pediatric Intensive Care Unit (PICU) of a tertiary referral pediatric hospital. RESULTS: The clinical diagnoses included neurology (35%), neonatology (16%), hematology (11%), autoimmune disorders (11%) immunodeficiency disorders (11%), infections other than neonatal sepsis (9%) and cardiology (6.5 %). The indications for IVIG use had an Evidence category Ia / Ib in 62 % of cases whereas the other 38 % had level II and III evidence. Choice of IVIG as a therapeutic option was based on failure of other treatment options to achieve response in 46.5%, lack of alternative treatment options 15.5 % and the need for urgent response in 38 %. Adverse events, duration and doses are reported. CONCLUSION: IVIG use is governed by availability of alternative options and the need for urgent response in critically ill children. Guidelines should be issued based on locally available treatment options and their cost effectiveness.

Isolation of poliovirus shedding following vaccination in children with antibody deficiency disorders.

INTRODUCTION: Prolonged excretion of oral poliovirus may occur in primary antibody deficiency states. Those patients who persistently excrete the virus may pose the risk of aiding viral propagation in the environment. This study therefore aimed to identify the potential for prolonged poliovirus shedding by patients diagnosed with congenital antibody deficiency disorders. METHODOLOGY: A cohort of children later diagnosed with antibody deficiency disorders was included in the study. Patient history was taken for each participant, with emphasis on vaccination data. Laboratory investigations included immunoglobulin profiles and stool sample collection at one month intervals from each patient, with follow-up for six months. The virus isolates were detected using enzyme-linked immunosorbent assay (ELISA) and molecular reverse transcription polymerase chain reaction (RT-PCR) techniques. RESULTS: On the initial sample screens, one patient revealed excretion one for Sabin-like strain 1 (SL1) and one patient revealed excretion for Sabin like strain 2 (SL2). Only one patient continued to shed the virus (SL1) on three successive samples and on follow-up. There was no correlation between the level of immunoglobulins and duration of virus shedding. CONCLUSION: The study demonstrates the low occurrence of prolonged vaccine polioviruses shedding in a group of children exposed to a live vaccine.

Hyperammonemia in the pediatric emergency care setting.

UNLABELLED: Hyperammonemia is encountered frequently in acutely ill children presenting for emergency care with altered levels of consciousness (ALOC). Ammonia production, metabolism, and excretion are affected by different variables. Hyperammonemia may be a transient state or may signify more grave etiologies as inborn errors of metabolism. Levels of ammonia are also affected by proper sampling technique, transport, and analysis. OBJECTIVES: To determine the level of ammonia in acutely ill children with ALOC, identify causes of hyperammonemia, and correlate levels with illness severity and morbidity. DESIGN: Observational study. SETTING: Emergency department at Cairo University Specialized Paediatric Hospital. METHODS: Fifty cases of acutely ill pediatric patients with ALOC who presented to the emergency department were included in the study from 2008 through 2009. Emergency department patients (n = 20) with known diseases that may induce hyperammonemia were excluded. Patients were subjected to detailed history taking with emphasis on factors affecting ammonia levels and thorough clinical examination. A cohort group of age- and sex-matched children acted as a control group. RESULTS: The measured blood ammonia level ranged between 13 and 265 µmol/L, with a mean level of 95 µmol/L. Sixty percent of the children with ALOC had ammonia levels of greater than 75 µmol/L, with levels greater than 200 µmol/L seen in 6% of the studied sample. The study demonstrated a highly significant statistical difference between children with ALOC and control groups.There was no correlation between blood ammonia level and age. Correlations of ammonia levels were also conducted in comparison with etiological diagnoses and laboratory parameters with no statistical significance.There was no statistical significance between ammonia level and duration of illness, Sequential Organ Failure Assessment score, or Glasgow Coma Scale score/Morray Scale score. CONCLUSIONS: Clinicians should consider testing children with ALOC for hyperammonemia, provided that a clear understanding of its metabolism and factors controlling it are understood. Proper sampling must be ensured. Mild elevations of ammonia levels are fairly common, but exceedingly high levels should raise concern and may require further evaluation.