Whole-exome sequencing of T- B+ severe combined immunodeficiency in Egyptian infants, JAK3 predominance and novel variants.

Severe combined immunodeficiency (SCID) is fatal if not treated with immune reconstitution. In Egypt, T- B+ SCID accounts for 38·5% of SCID diagnoses. An accurate genetic diagnosis is essential for choosing appropriate treatment modalities and for offering genetic counseling to the patient's family. The objectives of this study were to describe the clinical, immunological and molecular characteristics of a cohort of twenty Egyptian patients with T- B+ SCID. The initial diagnosis (based on clinical features and flow cytometry) was followed by molecular investigation (whole-exome sequencing). All patients had the classic clinical picture for SCID, including failure to thrive (n = 20), oral candidiasis (n = 17), persistent diarrhea (n = 14), pneumonia (n = 13), napkin dermatitis (n = 10), skin rash (n = 7), otitis media (n = 3) and meningitis (n = 2). The onset of manifestations was at the age of 2·4 ± 1·6 months and diagnosis at the age of 6·7 ± ·5 months, giving a diagnostic delay of 4·3 months. JAK3 gene variants were most frequent (n = 12) with three novel variants identified, followed by IL2Rγ variants (n = 6) with two novel variants. IL7Rα and CD3ε variants were found once, with a novel variant each. T- B+ NK- SCID accounted for approximately 90% of the Egyptian patients with T- B+ SCID. Of these T- B+ NK- SCID cases, 60% were autosomal recessive syndromes caused by JAK3 mutations and 30% were X-linked syndromes. It might be useful to sequence the JAK3 gene (i.e. targeted Sanger sequencing) in all T- B+ SCID patients, especially after X-linked SCID has been ruled out. Hence, no more than 10% of T- B+ SCID patients might require next-generation for a molecular diagnosis.

Phenotypical heterogeneity in RAG-deficient patients from a highly consanguineous population.

Mutations affecting recombination activation genes RAG1 and RAG2 are associated with variable phenotypes, depending on the residual recombinase activity. The aim of this study is to describe a variety of clinical phenotypes in RAG-deficient patients from the highly consanguineous Egyptian population. Thirty-one patients with RAG mutations (from 28 families) were included from 2013 to 2017. On the basis of clinical, immunological and genetic data, patients were subdivided into three groups; classical T- B- severe combined immunodeficiency (SCID), Omenn syndrome (OS) and atypical SCID. Nineteen patients presented with typical T- B- SCID; among these, five patients carried a homozygous RAG2 mutation G35V and five others carried two homozygous RAG2 mutations (T215I and R229Q) that were detected together. Four novel mutations were reported in the T- B- SCID group; three in RAG1 (A565P, N591Pfs*14 and K621E) and one in RAG2 (F29S). Seven patients presented with OS and a novel RAG2 mutation (C419W) was documented in one patient. The atypical SCID group comprised five patients. Two had normal B cell counts; one had a previously undescribed RAG2 mutation (V327D). The other three patients presented with autoimmune cytopaenias and features of combined immunodeficiency and were diagnosed at a relatively late age and with a substantial diagnostic delay; one patient had a novel RAG1 mutation (C335R). PID disorders are frequent among Egyptian children because of the high consanguinity. RAG mutations stand behind several variable phenotypes, including classical SCID, OS, atypical SCID with autoimmunity and T- B+ CID.

Chronic granulomatous disease: review of a cohort of egyptian patients

BACKGROUND: Chronic granulomatous disease (CGD) is an inherited disease that results from a defect in the phagocytic cells of the immune system. It is caused by defects in one of the major subunits of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. The clinical presentations of CGD patients are heterogeneous. OBJECTIVES: This is the first report from Egypt discussing clinical and laboratory data of twenty-nine patients (from 26 families) with CGD from a single tertiary referral centre. RESULTS: There were twenty male and nine female patients. The consanguinity rate was 76% (19/25). Their age of diagnosis ranged from 2 to 168 months with a mean of 52.8 months ± 49.6 SD. The most common manifestations were abscesses in 79.3% (deep organ abscesses in 37.9% of patients), followed by pneumonia in 75.8% and gastrointestinal symptoms in 27.5%. Rare but fatal complications were also reported among patients as one patient developed haemophagocytic lymphohistiocytosis (HLH) syndrome. Although X linked-CGD universally constitutes the most common pattern of inheritance; only 6 of our patients 6/25 (24%) belonged to this group with a Stimulation Index (SI) of 1-5, and confirmed by carrier pattern of their mothers. Mothers were not available for testing in four male children. Nineteen patients (76%) had autosomal recessive patterns; ten males and nine females patients based on having abnormal SI, positive history of consanguinity and their mothers showing normal SI. CONCLUSION: Increasing the awareness of physicians about symptoms of CGD may lead to earlier diagnosis of the disease, thus enhancing proper management and better quality of life

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Chronic granulomatous disease: Review of a cohort of Egyptian patients.

BACKGROUND: Chronic granulomatous disease (CGD) is an inherited disease that results from a defect in the phagocytic cells of the immune system. It is caused by defects in one of the major subunits of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. The clinical presentations of CGD patients are heterogeneous. OBJECTIVES: This is the first report from Egypt discussing clinical and laboratory data of twenty-nine patients (from 26 families) with CGD from a single tertiary referral centre. RESULTS: There were twenty male and nine female patients. The consanguinity rate was 76% (19/25). Their age of diagnosis ranged from 2 to 168 months with a mean of 52.8 months ± 49.6 SD. The most common manifestations were abscesses in 79.3% (deep organ abscesses in 37.9% of patients), followed by pneumonia in 75.8% and gastrointestinal symptoms in 27.5%. Rare but fatal complications were also reported among patients as one patient developed haemophagocytic lymphohistiocytosis (HLH) syndrome. Although X linked-CGD universally constitutes the most common pattern of inheritance; only 6 of our patients 6/25 (24%) belonged to this group with a Stimulation Index (SI) of 1-5, and confirmed by carrier pattern of their mothers. Mothers were not available for testing in four male children. Nineteen patients (76%) had autosomal recessive patterns; ten males and nine females patients based on having abnormal SI, positive history of consanguinity and their mothers showing normal SI. CONCLUSION: Increasing the awareness of physicians about symptoms of CGD may lead to earlier diagnosis of the disease, thus enhancing proper management and better quality of life.

Epidemiology 1, 2, 3: origins, objectives, organization, and implementation.

This supplement is a report on the Epidemiology 1, 2, 3 (EPI 1, 2, 3) investigation, its origins, evolution, and findings that were carried out over a period beginning in 1990 and ending in 1994 in Egypt. The large scope and size of the study, the largest to date on schistosomiasis in Egypt, was a rationale for publishing a supplement to document EPI 1, 2, 3 methods and results collectively in sufficient detail to serve as a reference for planning, designing, and analyzing future epidemiologic studies and evaluation of schistosomiasis control in Egypt. The 3 objectives of EPI 1, 2, 3 were to 1) determine the changing patterns of Schistosoma haematobium and S. mansoni, 2) investigate factors contributing to differences between villages in the Nile Delta, Middle Egypt, and Upper Egypt, and 3) investigate risk factors for morbidity. The objectives were addressed using standardized techniques, stool and urine examinations, clinical examinations (including abdominal ultrasound), and questionnaires on a selected sample of the populations of selected villages in 9 governorates in Egypt.

The epidemiology of schistosomiasis in Egypt: summary findings in nine governorates.

Health questionnaires and parasitologic examinations of urine and stool were evaluated from a stratified random sample of 89,180 individuals from 17,172 households in 251 rural communities in 9 governorates of Egypt to investigate the prevalence of, risk factors for, and changing pattern of infection with Schistosoma sp. in Egypt. A subset, every fifth household, or 18,600 subjects, had physical and ultrasound examinations to investigate the prevalence of and risk factors for morbidity. Prevalence of S. haematobium in 4 governorates in Upper Egypt in which it is endemic ranged from 4.8% to 13.7% and averaged 7.8%. The geometric mean egg count (GMEC) ranged from 7.0 to 10.0 ova/10 ml of urine and averaged 8.1. Age stratified prevalence of infection peaked at 15.7% in the 10-14-year-old age group and decreased to 3.5-5.5% in all groups more than 25 years of age. Age-stratified intensity of infection peaked at approximately 10.0 ova/10 ml of urine in the 5-14-year-old age groups and was about half that in all groups more than 25 years of age. Males had higher infection rates and ova counts than females in all age groups. Schistosoma mansoni was rare in Upper Egypt, being consequential in only Fayoum, which had a prevalence of 4.3% and an average intensity of infection of 44.0 ova/g of stool. Risk factors for S. haematobium infection were male gender, an age <21 years old, living in smaller communities, exposures to canal water; a history of, or treatment for, schistosomiasis, a history of burning micturition or blood in the urine, and reagent strip-detected hematuria or proteinuria. The more severe grades (II and III) of ultrasonography-detected periportal fibrosis (PPF) were rare (15 of 906) in these schistosomiasis haematobia-endemic governorates. Risk factors for morbidity (ultrasonography-detected urinary bladder wall lesions and/or obstructive uropathy) were similar to those for infection, with the exception that risk progressively increased with age. Subjects with active S. haematobium infections were 3 times as likely as those without active S. haematobium infections to have urinary tract morbidity. The prevalence of S. mansoni in 5 governorates in Lower Egypt, where it is endemic, ranged from 17.5% to 42.9% and averaged 36.4%. The GMEC ranged from 62.6 to 93.3 eggs/g of stool and averaged 81.3. Age-stratified prevalence of infection peaked at 48.3% in the 15-19-year-old age group, but averaged 35-45% in all groups more than 10 years of age. The intensity of infection was highest in the 10-14-year-old age group, and showed a range of 70-85 eggs/g of stool in those > or =5 years of age. Males had higher infection rates and ova counts than females in all age groups. Schistosoma haematobium was rare in these governorates; Ismailia (1.8%) had the highest infection rate. Risk factors for S. mansoni were male gender, an age >10 years old, living in smaller communities, exposures to canal water, a history of, or treatment for, schistosomiasis or blood in the stool, detection of splenomegaly by either physical examination or ultrasonography, and ultrasonography-detected PPF. The more severe grades (II and III) accounted for 463 (13.3%) of the 3,494 having ultrasonography-detected PPF. Risk factors for morbidity (ultrasonography-detected PPF) were similar to those for infection except that inhabitants of smaller communities were not at increased risk. Active S. mansoni infection increased the odds ratio (OR) of having PPF by 1.37. In comparison with others with normal-size livers, subjects having hepatic enlargement in either the midclavicular line or the midsternal line detected by physical examination or ultrasonography had a reduced risk (ORs = 0.64-0.72) of PPF. The prevalences of lesions detected by ultrasonography were 23.7% for enlargement of right lobe of the liver, 11.3% for enlargement of left hepatic lobe, 20.6% for splenomegaly, and 50.3% for PPF. Schistosoma mansoni has almost totally replaced S. haematobium in Lower E

Detection of circulating antigens in patients with active Schistosoma haematobium infection.

An antigen-capture ELISA using monoclonal antibody (MAb) 128C3/3/21 was used to detect circulating parasite-derived antigens in the sera of patients actively infected with Schistosoma haematobium (31 males and four females, 5-25 years of age). The assay had a sensitivity of 100% (35 of 35 patients with antigen levels > 80 ng/ml) and a specificity > 99%. We used this ELISA to monitor antigenemia before treatment and one, three, and six months after treatment with a single oral dose of praziquantel (PZQ) (60 mg/kg in 20 patients or 40 mg/kg in 15 patients) and compared our findings with those indicated by other measures of disease progression. Circulating antigen levels decreased drastically after PZQ treatment (P < 0.001), with a significantly higher decrease occurring after treatment with 60 mg/kg than with 40 mg/kg. Although the mean antigen level was still significantly reduced (P < 0.001) at six months after treatment, 16 patients remained antigen-positive after six months, and nine had increased levels of antigenemia, reflecting reinfection in six patients and persistence of infection in another. We observed a correlation (r = 0.6, P < 0.01) between the level of circulating antigen and the intensity of infection as measured by egg count. We also found a direct relationship (P < 0.001) between antigen level and the severity of the disease as monitored by ultrasonography. We conclude that our ELISA may be a useful adjunct to other methods, such as ultrasonography, for monitoring the course of S. haematobium infection and treatment.

The USAID/Government of Egypt's Schistosomiasis Research Project (SRP).

Schistosomiasis is the major public health problem in rural Egypt, with almost six million Egyptians infected as at mid-1996. In 1983, the prevalence of schistosomiasis in rural Egypt was greater than 50%, but a ten-year campaign of diagnosis and treatment has reduced the prevalence and intensity of infection. Parallel to this campaign, the government of the USA has funded a research project to examine all aspects of schistosomiasis with a view to improving the control strategy. As outlined here by Taha El Khoby, Nabil Galal and Alan Fenwick, after almost ten years, the project's achievements include: assisting WHO in its attempts to develop a vaccine against schistosomiasis, developing a suspension formulation of praziquantel suitable for young children, and establishing a unit to monitor reports of resistance to praziquantel. In addition, a large epidemiological study has established the extent of the problem in Egypt. Slow-release formulation of niclosamide, health education material for use on TV, dipstick diagnostic tests, and evaluation of ultrasound as a diagnostic tool have also been achieved. As the project closes, Egypt is left with an effective strategy for the control of schistosomiasis and several back-up tools for use in the event of development of resistance to praziquantel. The Ministry of Health and Population has the basis of a Geographical Information System (GIS) unit and the country has a trained and equipped scientific community capable of biomedical research, and almost 100 scientific papers published on their work.

Transmission of circulating schistosomal antigens from infected mothers to their newborns.

Two previous reports have appeared in the literature regarding tansplacental transfer of Schistosoma mansoni antigens which raised the question of its reality. In a previous study the senior author, and others (1992 & 1997) detected circulating antigens of S. mansoni and S. haematobium in infected patients, using monoclonal antibodies 128C3, with a very high sensitivity using ELISA. This work tried to answer the question of antigen transfer possibility using a high sensitive assay in 50 mothers and their newborns at birth and 1, 3, and 6 months after delivery. The assay used in the present work could detect circulating S. mansoni antigens in 45 infected mothers (90%) with active S. mansoni infection. A significant direct increase in mean antigen levels was found with the intensity of infection evaluated by egg counting (p < 0.01). The clinical stage of the diseased mothers was apparently unrelated to the ELISA test values as no significant relations were observed. Positive antigen levels were detected in 33 newborns (66%) of the 45 positive antigen mothers, then the percentage positivity was directly decreased with the advancement of age as only 5 infants (10%) had positive antigen levels compared to 0% at 6 months of age. A positive correlation between newborn serum antigen concentration and concentration of antigen in sera of their mothers was obtained. This work answers some of the questions concerning the ability of the used monoclonal to detect antigens in newborns and the possibility of antigen transfer through the placenta alone or incorporated in immune complexes forms. This work clarifies the time of antigen disappearance from the circulation.

Parasitic causes of hepatomegaly in children.

Three hundred children with hepatomegaly were selected. They were subjected to full clinical and laboratory examinations. Also serum samples were examined to detect IgG using ELISA against SEA, chromatography purified hydatid cyst antigen, commercially available Toxoplasma antigen, partially purified adult Fasciola antigen and second-stage larvae Toxocara canis antigen. IFAT was used to detect IgG against Toxoplasma and T. canis. A commercially available IHAT kit for leishmaniasis was used. Based on immunological assays, 125 cases were suffering from various parasitic infections. Thirty cases with schistosomiasis (10%), 26 cases fascioliasis (8.7%), 18 toxocariasis (6%), 35 toxoplasmosis (11.7%), 3 cases hydatidosis (1%) and 13 cases mixed parasitic infections. No parasitic causes could be found in 175 cases (58.3%). Moderate or marked hepatomegaly favours the presence of schistosomiasis. Whereas, most cases with other parasites and those with non-parasitic infections fall in the category of mild hepatic enlargement. There was no associated splenomegaly in cases with Fasciola, Toxocara, hydatid disease and/or the non-parasitic group. Most of hepatomegalic cases with non-parasitic causes were found to be associated with fever (88.5%). Fever was found in nearly 50% of cases with either Toxoplasma or Toxocara infections. Mild eosinophilia was found in all cases with parasitic causes. Only 24 cases of non-parasitic group (13.7%) had easinophilia. Moderate and high eosinophilia were found in cases with fascioliasis and toxocariasis. Cases with fascioliasis had a statistically significant increase in enzymes activities specially alkaline phosphatase. It was concluded that parasitic infections should be considered as an important cause of liver enlargement in children. Serological methods using purified antigenic fractions are an important tool for diagnosis.

Immune response to Schistosoma mansoni infections in inbred rats. VII. Resistance is contingent on OX-8(+)-regulated high affinity IL-2 receptor-bearing W3/25+ lymphocytes but not on IL-4-dependent cells.

These studies assess the roles of subpopulations of T lymphocytes in resistance to Schistosoma mansoni. CDF rats were depleted of the T cell subpopulation bearing the high affinity IL-2R by in vivo treatment with ART18+ mAb or of soluble IL-4 by treatment with 11B11 mAb. The development of parasites, the expression of resistance after sensitization, and the intensity of delayed type hypersensitivity (DTH), Ag-mediated blast transformation (AMBT), IgG2a, passive cutaneous anaphylaxis, and IgE-mediated antibody-dependent cell-mediated cytotoxicity responses against S. mansoni or control Ag were ascertained. Isolated T cell subpopulations were assessed in vivo and in vitro for effects upon the protective Ir. Depletion with ART18 mAb suppressed the development of W3/25+ helper-inducer cells and resulted in the initial survival of more worms, decreased resistance to challenge after initial sensitization, decreased IgG2a and IgE antibody, AMBT, and DTH reactivity against schistosome Ag. Depletion with ART18 did not prevent the development of OX8+ (T suppressor) cells. Depletion with 11B11 mAb led to insignificant changes in initial parasite survival and resistance to challenge; had no effect on IgG2a antibody, AMBT, or DTH; but profoundly suppressed the IgE responses against the parasite. Protective immunity to S. mansoni in rats is dependent upon IL-2R-bearing T lymphocytes and regulated by OX8+ cells but not absolutely contingent upon IL-4 or the IgE response.

Resistance in murine schistosomiasis is contingent on activated IL-2 receptor-bearing L3T4+ lymphocytes, negatively regulated by Lyt-2+ cells, and uninfluenced by the presence of IL-4.

These studies assess the roles of subpopulations of T lymphocytes in inducing and modulating resistance to Schistosoma mansoni. C57BL/6 mice were depleted in vivo of L3T4+, Lyt-1+, Lyt-2+, IL-2R+ cells, or IL-4 by administration of appropriate mAb. Resistance and various correlative parameters of the immune response were studied in normal, depleted, and congenitally athymic mice. Depletion of T lymphocytes by anti-L3T4 or anti-IL-2R mAb reduced the development and expression of resistance, IgG2a and IgE antibody formation, and delayed type hypersensitivity reactivity against schistosome Ag. Depletion with anti-IL-4 antibody led to profound suppression of IgE-eosinophil-mediated antibody-dependent cell-mediated cytotoxicity and passive cutaneous anaphylaxis responses against the parasite and no effect on IgG2a antibody, Ag-mediated blast transformation, or resistance. Depletion of Lyt-2+ cells produced augmented development and expression of resistance and an increase in the immunological parameters of anti-schistosome reactivity. These studies suggest that protective immunity to S. mansoni in mice, induced by irradiated cercariae, is dependent on L3T4+, IL-2R+ lymphocytes and negatively regulated by Lyt-2+ cells. IL-4 does not appear to be essential for the development of resistance but is essential for the IgE response to the parasite.

The regulation of resistance to Schistosoma mansoni by auto-anti-idiotypic immunity. III. An analysis of effects on epitopic recognition, idiotypic expression, and anti-idiotypic reactivity at the clonal level.

Auto-anti-idiotypic mechanisms can regulate the protective immune response against Schistosoma mansoni. Anti-idiotypic responses were stimulated by immunization of mice either with nonspecifically induced lymphoblasts, produced with Con A, or with Ag-induced lymphoblasts bearing specific idiotypic receptors. The effect of the induced anti-idiotypic response upon clonotypic cellular reactivity was assessed in vitro through the suppression of antigen-mediated blast transformation by cloned T cells and in vivo by suppression of resistance to S. mansoni and delayed-type hypersensitivity responses against specific Ag. Differential regulation of humoral immune responses was studied at the levels of specific epitopic recognition, the expression of specific Id, and the production of anti-idiotypic responses directed against mAb bearing specific Id. Anti-idiotypic sensitization resulted in variable (10 to 90%) suppression of the immune response to discrete antigenic epitopes, the expression of specific idiotypic phenotypes, and anti-idiotypic, antiparatopic responses against T cell clonotypes and antibody idiotypic phenotypes. In vitro admixture and in vivo challenge studies resulted in consonant differential suppression. Thus idiotypic regulation can mold the fine specificities of the protective immune response to S. mansoni at the clonal level and may provide an approach to optimize the expression and assessment of resistance.