Approach to Primary Immunodeficiency.pptx

Approach to primary immunodeficiency..

1. APPROACH TO PRIMARY IMMUNODEFICIENCY DISEASES (PID) PRESENTER: Dr R K Shwetabh MODERATOR: Dr Abhijeet Roy(Asst Prof,Dept of Neonatology,SGPGIMS )

2. Specific Learning Objectives Introduction and Basic Immunology Classification When to suspect Evaluation Management

3. Introduction Primary immunodeficiency disorders are a heterogenous group of inherited disorders which affect different components of immune system. More than 150 different disorders which have been described till date M any patients remain undiagnosed or are diagnosed too late with severe consequences. Around 6 million people live with a PID worldwide but between 70-90% are undiagnosed. Practical approach to diagnosis and management of PID-Rossouw TM et al,2018 Diagnostic Approach to Primary Immunodeficiency disorders-M Madkalkar et al,2013

4. Immunity INNATE/NATURAL IMMUNITY Anatomical and physical barrier Phagocytic cells( neutrophils & macrophages) NK cells Complement system ACQUIRED/ADAPTIVE IMMUNITY Active Passive ‘ Humoral immunity-B cell mediated Cell mediated-T cell mediated

5. B lymphocytes Antibody production- Neutralisation of microbes & toxins Opsonisation & phagocytosis Complement mediated lysis T helper lymphocytes Cytokine production (Th1=IL2, INF γ ; Th2= IL4, IL5, IL13; Th17= IL17,IL 22) Activation of macrophages, killing of ingested microbes Inflammation Activation (proliferation & differentiation of B & T lymphocytes) Cytotoxic T lymphocyte Killing of infected cell Natural killer cell Killing of infected cell (antibody independent cytotoxicity) Function

6. Function Neutrophils Inflammation Anti microbial property Phagocytosis,degranulation,generation of NET Macrophages Phagocytosis Leucocyte recruitment eg CXCL8,IFN 1 Activation of vascular endothelium-TNF alpha Leucocyte activation-TNF alpha,IL 6,IL 12,IL 1 beta Tissue repair Complement System Antiviral property Immunological membrane damage( cytolysis,bacteriolysis ) Hypersensitivity reaction

7. Neonates- What is different?

8. Innate Immunity Term neonate immune system-Anatomically complete but antigenically inexperienced. Newborns have reduced neutrophil production as well as reduced qualitative function. Macrophages and NK cells have diminished cytotoxicity. Complement system do not reach adult levels until 12-18 months of life. Preterm neonates-Fragile skin,moderate –severe hypogammaglobinemia,lower lymphocyte count and plasma complement. Primary Immunodeficieny diseases in the newborn,Oner Ozdemir,2021

9. Humoral Immunity Neonate is dependent on passively acquired maternal immunoglobulin G ( IgG ), which passes through the placenta predominantly during the third trimester of gestation Preterm infants at <34 weeks of gestation have significantly less serum IgG . Maternally derived IgG may wane faster with repeated phlebotomy, surgery and infection. IgA, IgM and IgE do not pass through the placenta.

10. Physiologic Hypo gammaglobinemia of Infancy(PHI) Maternal transferred IgG existent at birth and disappears over 3-6 months Steady maturation of B cells to plasma cells occur Leads to PHI IgG level < 400 mg/dl 3-6 months Premature neonates-PHI earlier,intense and persists longer

11. Cellular Immunity Newborn infants have decreased pro-inflammatory/T-helper cell type 1 (Th1) cytokine release and cell-mediated immunity . CD4 cells have decreased activity to produce IFN gamma. T-cells have decreased effective cytotoxicity Physiologically diminished T-cell function leaves the neonate susceptible to both infection and autoimmunity. ALC of <2500 cells/mL suggests a T-cell and/or B cell deficiency . Primary Immunodeficieny diseases in the newborn,Oner Ozdemir,2021

12. Immunodeficiency Disease Classification PRIMARY IMMUNODEFICIENCY DISEASES (PIDs) SECONDARY IMMUNODEFICIENCIES Severe acute malnutrition HIV infection Measles Immunosuppressive therapy Nephrotic syndrome Malignancy (lymphoma/leukemia) Diabetes, renal failure, severe liver disease etc Defects in innate immunity Defects in acquired immunity

13. Primary Immunodeficiency Disorder Congenital disorders caused by intrinsic molecular defect that alters development or function of immune system Incidence- 1:10000/live births Most Common –Selective IgA deficiency(1:500 incidence) Male> Female(Infant age group) Majority (70-90%) remain undiagnosed PID- higher in genetically isolated populations Practical approach to diagnosis and management of PID-Rossouw TM et al,2018 Diagnostic Approach to Primary Immunodeficiency disorders-M Madkalkar et al,2013

14. Number of patients with various PIDs at PGIMER Chandigarh (Jan 1990- March 2017)

15. When to suspect?

16. 10 Warning signs for suspicion of PIDs (Jeffery Modell foundation & ESID) > 4 new ear infections within 1 year. > 2 serious sinus infections within 1 year. > 2 deep seated infections including septicaemia. > 2 pneumonias within 1 year. > 2 months on antibiotics with little effect. Failure of an infant to gain weight or grow normally Need for intravenous antibiotics to clear infections. A Family history of PID. Recurrent deep skin or organ abscesses. Persistent thrush in mouth or fungal infection in skin. - McCusker and Warrington Allergy, Asthma & Clinical Immunology 2011 90% cases

17. CLASSIFICATION

18. Classification of primary immunodeficiency diseases {International union of immunological societies (IUIS) classification} Combined T and B cell immunodeficiencies Combined immunodeficiencies with associated or syndromic features Predominantly antibody deficiency Diseases of immune dysregulation Congenital defects of phagocyte number and function Defects in innate immunity Autoinflammatory disorders Complement deficiencies Phenocopies of PID

19. PID category Examples Combined T & B cell immunodeficiencies Severe combined immunodeficiency(SCID) Combined immunodeficiencies with associated or syndromic features Ataxia telangiectasia DiGeorge syndrome Wiskott Aldrich syndrome Hyper IgE syndrome Predominant antibody deficiencies X linked agammaglobulinemia Common variable immunodeficiency Hyper IgM syndrome Selective IgA deficiency IgG subclass deficiency Transient hypogammaglobulinemia of infancy Diseases of immune dysregulation Chediak Higashi syndrome Familial hemophagocytic lymphohistiocytosis Autoimmune Lymphoproliferative syndromes IPEX (Immune dysregulation, polyendocrinopathy, enteropathy, X linked syndrome) APECED (Autoimmune polyendocrinopathy, candidiasis, ectodermal dysplasia)

20. PID category Examples Defects of phagocytic number, function or both Severe congenital neutropenia Cyclic neutropenia Leucocyte adhesion deficiency Chronic granulomatous disease Defects in innate immunity Mendelian susceptibility to mycobacterial disease (MSMD) Chronic mucocutaneous candidiasis Autoinflammatory disorders Familial Mediterranean fever Familial cold autoinflammatory syndrome Neonatal onset multisystem inflammatory disease (NOMID) Complement deficiencies Deficiencies of early (C1q, C1r, C2, C4) and late complement pathway components (C5, C6, C7, C8, C9) C1 inhibitor deficiency Phenocopies of PID(no genetic defect/somatic mutation) Antibodies to IL-7 or IL-22 Antibodies to C1 inhibitor

21. Evaluation of Suspected immunodeficiency HISTORY – “recurrent infections” hallmark of immunodeficiency SYMPTOMS PHYSICAL EXAMINATION INVESTIGATIONS- baseline and advanced tests

22. Relevant Points in History - Age of initiation of infections- Onset before 6 month of age - T cell defect Onset between 6-12 months – Combined B & T cell defect or B cell defect Onset later than 12 month- B cell defect or secondary immunodeficiency -Frequency, duration, severity of infections & response to antibiotics -Sites & number of infections (ear discharge, recurrent/chronic diarrhea, boils, deep seated abscesses, skin lesions, oral thrush)

23. Points & Pointers In History Significant points Disease FTT,Chronic diarrhoea SCID Eczematous rash Wiskott Aldrich Syndrome Delayed separation of umbilical cord LAD Albinism Chediak Higashi Severe dermatitis Omenn Syndrome History Points Antenatal history-History of sibling death Similar illness in family History of consangunity Previous hospitalization Development history Immunization history

24. Physical examination Examine potential sites of infection- throat, ears, sinuses, oral cavity, diaper area for candidiasis, skin & appendages Presence or absence of lymphoid tissue, organomegaly Facial features Signs eg clubbing for respiratory infections Anthropometry Specific clinical features according to different diseases

25. Signs that could point to PID Examination Finding General examination Clubbing, Absence of tonsil, lymph node Head to toe Bleeding diathesis Skin and appandages hypopigmented hair, seborrhea eczema, severe dermatitis, neonatal exudative erythroderma, poor wound healing, scarring, angioedema (without urticaria) Oral cavity severe gingivostomatitis, recurrent aphthous ulcers Eyes retinal lesions, telangiectasias Dysmorphism Congenital cardiac disease

26. CLINICAL FEATURES DISORDERS Diarrhea , failure to thrive, life threatening infections Severe combined immunodeficiency (SCID) Severe dermatitis, erythroderma, hepatosplenomegaly, lymphadenopathy Omenn syndrome Ocular telangiectasia, ataxia, recurrent sinopulmonary infections Ataxia telangiectasia Hypocalcemic tetany, congenital heart defect, unusual facies, low set ears, development delay Di George syndrome Recurrent infections, eczema, bleeding manifestations, bloody diarrhea, persistent thrombocytopenia, small platelets Wiskott Aldrich syndrome Recurrent abscesses of skin, lungs, joints, pulmonary pneumatoceles, pruritic dermatitis, coarse facies, hyper eosinophilia, eczema AD Hyper IgE syndrome Eczema, extensive molluscum contagiosum, recurrent abscesses, asthma, atopy, food allergy, hyper eosinophilia AR Hyper IgE syndrome Combined immunodeficiencies with associated or syndromic features Combined T & B cell immunodeficiencies

27. CLINICAL FEATURES DISORDERS Abscess, lymphadenopathy, pneumonia, osteomyelitis IPEX Oculocutaneous albinism, neurologic changes, lymphadenopathy Chediak Higashi syndrome Abscess, lymphadenopathy, pneumonia, osteomyelitis Chronic granulomatous disease Delayed separation of umbilical cord,recurrent infection Leucocyte Adhesion Defect Chronic gingivitis, recurrent aphthous ulcers & skin infections, severe neutropenia Congenital neutropenia Defects in innate immunity Disseminated mycobacterial infection/ BCGiosis / recurrent salmonella Mendelian susceptibility to mycobacterial diseases Complement deficiencies Recurrent Neisseria meningitis C5, C6, C7 or C8 deficiency Diseases of immune dysregulation Defects of phagocytic number, function or both

28. Clues to presence of PID PID AGE AT ONSET OF INFECTION COMPLICATIONS ORGANISMS SPECIAL FEATURES Predominant T cell defect Early onset, before 6 month of age Systemic viral infections, gastroenteritis, extensive mucocutaneous candidiasis, failure to thrive, protracted diarrhea Bacteria : common gram positive & gram negative bacteria, mycobacteria Viruses : All, CMV, EBV, parainfluenza 3, varicella, adenovirus, enterovirus Fungi: candida, Pneumocystis carinii Parasites: toxoplasma, cryptosporidium -Graft vs host disease (maternal T cells or non- irradiated blood transfusion) -Clinical infection due to live attenuated vaccines (postvaccination disseminated BCG or varicella, diarrhea post rotavirus vaccine) -Absent lymphoid tissue -Absent thymic shadow on CXR - M Madkaikar , A Mishra, K Ghosh, Indian Pediatrics 2013

29. PID AGE AT ONSET OF INFECTION COMPLICATIONS ORGANISMS SPECIAL FEATURES B CELL DEFECT Onset after 6 month of age, later childhood to adulthood Recurrent sinopulmonary infections, chronic GI symptoms, malabsorption, arthritis, enteroviral meningoencephalitis Bacteria: P neumococci, Streptococci, Staphylococci, H. influenzae, Campylobacter, Mycoplasma Viruses: E nterovirus Fungi and parasites : Giardia -Familial clustering of autoimmune disorders -Lymphoreticular malignancy - M Madkaikar , A Mishra, K Ghosh, Indian Pediatrics 2013

30. PID AGE AT ONSET OF INFECTION COMPLICATIONS ORGANISMS SPECIAL FEATURES PHAGOCYTE DEFECTS Early onset Skin: abscesses, impetigo, cellulitis Lymph nodes: suppurative adenitis Oral cavity: gingivitis, mouth ulcers, Liver/lung/brain abscesses, osteomyelitis Bacteria: Staphylococcus, Pseudomonas, Salmonella, Klebsiella, Nocardia, Serratia Fungi : Candida, Aspergillus -Delayed detachment of umbilical cord -poor wound healing COMPLEMENT DEFECTS Onset at any age Meningitis, arthritis, septicaemia, recurrent sinopulmonary infections Bacteria: pneumococci, Neisseria -Autoimmune disorders: SLE, vasculitis, dermatomyositis, scleroderma, glomerulonephritis -Hereditary angioedema

31. 1 st line investigations- Full blood count with differential count on leucocytes Lymphopenia (age related range)- T cell disorder or combined immunodeficiency (Birth < 3000/ μ l, 5-6 months up to 1 year < 4000/μl, adult < 1000/μl) Normal ESR rules out serious bacterial & fungal infection Neutropenia (ANC< 1500)- congenital or cyclic or aplastic Neutrophilia in absence of overt infection- LAD or CGD Abnormal leucocyte granules in PS - Chediak Higashi syndrome Thrombocytopenia, low platelet volume in male infant- WAS Ig A levels- screening test. Normal rules out T & B cell defect. Laboratory Approach to Patient With PID - Udayasankar S (2017) Approach to Immunodeficiency Disorders. J Pediatr Neonatal Care

32. Laboratory Approach to Patient With PID

33. Structured approach for immunological testing (Jeffery Modell foundation) Step 1 History, physical examination, weight & height Exclude HIV infection CBC & differential count Quantitative immunoglobulin levels (IgA, IgM, IgG, IgE) Step 2 Specific antibody production (pre & post immunisation titres) IgG subclass analysis Step 3 Lymphocyte subsets (CD3, CD4, CD19, CD16/56) Complement screen (CH50/ CH100) Neutrophil oxidation burst (if indicated) Lymphocyte proliferation studies (using mitogen & antigen stimulation) Step 4 Advanced testing guided by experienced clinician e.g. mutational studies

34. Flow cytometry Rapid and sensitive tool for diagnosis and classification of PIDs Numbers of T cells, B cells, and NK cells. Provide the percentage of naive (CD45RA1) versus memory (CD45RO1) T cells present. T-lymphocytes of maternal origin will be memory cells. Normal neonate T cells are approximately 90% naive.

35. TREC (T cell recombination excision circles ) T-cell receptor excision circles  (TRECs) are small circles of DNA created in  T-cells  during their passage through the  thymus  as they rearrange their  TCR  genes. Circular, non replicating pieces of DNA , Surrogate marker for naïve T cells Inability to detect TREC by quantitative reverse transcription polymerase chain reaction ( qRT -PCR)- test for new-born screening of SCID using dried blood spots. Only some of the children who have low T cells/ TREC will have SCID. Abnormal TREC Screen-idiopathic T-cell lymphopenia , prematurity-related immune immaturity, chylothorax , and genetic syndromes

36. KREC (Kappa—deleting recombining excision circles) C ircular DNA segments generated in B cells during their maturation in bone marrow .  Produced during rearrangement of the variable, diversity and joining domains (V(D)J recombination) of the B cell immunoglobulin kappa gene . Assay measuring TREC and KREC, along with beta-actin levels as a control for DNA quantity- simultaneous screening for T and B cell lymphopenia . After HCT, rise in KRECs reflects newly derived bone marrow B cells . Fail to predict development of antibody deficiencies later in life such as CVID.

37. Prenatal Diagnostic Techniques Family history is positive for SCID. Available DNA tests allow for the identification of mutations involving ADA, RAG1/RAG2, JAK3, γ C, IL-7 receptor and Artemis,. Chorionic villus sampling at 10 weeks’ gestation (or later) by amniocentesis, in families for whom the exact mutations have been established. Fetal blood sampling for fluorocytometric testing, mitogen responses, and enzyme levels can establish the diagnosis when DNA analysis is not available. Percutaneous umbilical blood sampling is performed to examine fetal blood for T-cell deficiency, as well as ADA enzyme levels

38. SUMMARY OF TREATMENT Supportive treatment Definitive treatment SCID or CIDs Ig replacement therapy, Antibiotic prophylaxis Antifungal prophylaxis Aggressive management of established infections Withhold all live vaccines BMT HSCT Gene therapy B cell disorders Ig replacement therapy, Antibiotic prophylaxis Antifungal prophylaxis Assessment of pulmonary status & function Gene therapy- potential future Innate disorders Antibiotic prophylaxis, Antifungal prophylaxis Cytokine replacement Vaccination BMT Gene therapy- potential future The mainstay of therapy for most PIDs involves preventing infection Interferon therapy has been shown to prevent infections in patients with CGD

39. Common PID Presenting in Neonatal Period Severe Combined Immunodeficiency Di George Syndrome Leucocyte Adhesion Defect Wiskott Aldrich Syndrome Chronic Granulamatous Disease Immunoregulatory disorder-HLH

40. Severe Combined immunodeficiency Diverse genetic mutation-Absent T cell and B cell function Previously known as Bubble baby disease David Vetter -12 yr old boy of Texas born in 1971 spend entire life of 12 years in sterile plastic bag. NK cell function may also get affected MC –X linked SCID(IL-2RG-CD132 deficiency)

41. Classification

42. Severe Combined Immunodeficiency T-B- SCID T-B-NK- Chondrosternal dysplasia, deafness (ADA deficiency) Granulocytopenia, thrombocytopenia, deafness (Reticular dysgenesis) T-B-NK+ RAG1/RAG2 Artemis deficiency Yes+ facial dysmorphism ?microcephaly no DNA ligase 4 T-B+ SCID T-B+NK- T-B+NK+ X-linked CD132- γ chain deficiency ( IL2,4,7,9,21) AR- CD132+ JAK 3 deficiency Distinctive Clinical features Yes+growth retardation Cernuounus Yes CORO1A deficiency No IL7R

43. Severe Combined Immunodeficiency CLINICAL FEATURES Early onset, persistent skin, respiratory or gut infection Chronic diarrhea , Failure to thrive Infections with opportunistic organisms (pneumocystis jiroveci Prolonged otitis media, invasive bacterial infections (pseudomonas or staphylococcus sepsis or pneumonia) Extensive, persistent superficial candidiasis Disseminated BCG infection or vaccine strain poliomyelitis Absent Tonsils,thymus,hypoplastic lymph nodes Graft verses host disease (GVHD)- inability to reject foreign lymphocytes acquired either from mother in utero or from un-irradiated blood transfusion

44. Severe Combined Immunodeficency investigations Profound lymphopenia T cells<1500 cells Hypogammaglobinemia Flow cytometry Management Isolation Leuco-reduced, irradiated transfusion Live vaccine contraindicated Antibiotic prophylaxis Immunoglobulin-Humoral defect HSCT ADA

45. OMENN SYNDROME Rare severe morphological manifestation in some SCID Hypomorphic mutation in SCID gene-RAG 1,2Aartemis or IL-7Ra Erythroderma Alopecia FTT Eosinophilia Treatment-HSCT

46. Di George Syndrome

47. Di George Syndrome Di George Syndrome Management: -Transplant of small epithelial cells into quadriceps -De novo production starts in 4-6 weeks Severe T cell lymphopenia Hypoparathyroidism Conotruncal congenital heart disease Rash GI symptom Hepatits Complete Atypical

48. Physical examination Failure to thrive Adverse reactions after live vaccines Common or unusual infections in an infant or young child Intractable diarrhea, viral infections Cutaneous findings Absence of thymus, lymphoid tissue Lab testing CBC & differential Lymphopenia Advanced tests Treatment Immunoglobulin replacement Bone marrow transplant Gene therapy CELLULAR OR COMBINED IMMUNE DEFECTS

49. Leucocyte Adhesion Defect (LAD ) Defective function of neutrophils, monocytes, T cells & NK cells Depressed chemotaxis, adherence & phagocytosis Present in first week of life with delayed umbilical cord separation & progressive perianal ulcers Lack of pus formation Deep seated infections of bone, respiratory & GI tracts Lab findings- neutrophilia & profound neutrophilic chemotactic defect

50. Leucocyte Adhesion Defect

51. Chronic Granulomatous disease Inherited defect of phagocyte NADPH enzyme complex Absence of NADPH oxidase- decreased/ absent oxidative burst & production of ROS Recurrent, potentially fatal bacterial or fungal infections Acute suppurative lymphadenitis, liver abscesses, osteomyelitis, arthritis, pneumonia, skin sepsis, perianal abscesses Infections with catalase positive organisms (Staphylococcus, Burkholderia, Aspergillus, Serratia) Disseminated fungal infection with osteomyelitis & hepatic involvement Non infectious granulomatous complications- IBD, restrictive lung defects, genitourinary obstruction, cutaneous granulomas (granulomatous infection occur due to failure to clear infection)

52. Chronic Granulomatous Disease

53. Chronic Granulamatous Disease Investigation a)DHR(Dihydro Rhodamine Test)-Using Flow Cytometry b)NBT test Treatment a)HSCT –Treatment of choice Prophylaxis-Cotrimoxazole and Itraconazole b)Steroids- Granulamatous Colitis MAS

54. skin History & Physical examination PHAGOCYTIC DEFECT Gastrointestinal tract lymphadenopathy musculoskeletal pulmonary Constitutional symptoms Laboratory tests CBC, Differential count, ESR, CRP Respiratory burst assay (NBT/DHR test) CD11/CD18 by flow cytometry Serum IgE levels Treatment Antibiotics Antifungal agents Bone marrow transplant Interferon gamma

55. Hemophagocytic Lymphohistiocytosis

56. Cases

57. Case 1 Term,Male,Birth weight-3kg History of MAS & HIE II Came at DOL33 to our hospital after referred from a pvt hospital Hepatosplenomegaly on examination Blood culture –K pneumoniae Received multiple antibiotics Immunodeficiency suspected Gene sequencing was done

58. Case 1

59. Case 2 Term,AGA,Male,Bwt-3.2 kg History of cyanosis since DOL2 Admitted to our hospital Diagnosed as CCHD(Truncus Arteriosus Type 1)with OS ASD and VSD Ventilated Baby also had gangliocapsular bleed Digeorge Syndrome suspected Expired on DOL 12

60. Report:

61. Case 3 33 week, Male, Bwt 2kg, AGA Histort of 3 rd degree consanguinity and 2 nd sibling death along with delayed separation of umbilical corf No history of delayed separation of umbilical cord in present baby Presented to us on DOL 45 with history of continuous high grade fever for 15 days History of NICU admission for 10 days at birth at a pvt hospital History of raised TLC count -62400,58700 and 54160 on 3consecutive days and hence referred to our hospital.

62. Case 3 Antibiotics were started and proper work up was done Baby developed umbilical swelling and discharge and TLC count were throughout in higher range Umbilical pus C/S-Citrobacter koseri

63. Case 3

64. Case 3

65. Case 3 CRP –Positive Peripheral smear-Normal Flow cytometry-Absent CD11/CD 18 expression LAD I -Diagnosed Baby referred for stem cell transplantation

66. Report

67. Case 4 39 week/2.8 kg/AGA/F/NVD/Non consanguineous marriage DOL 6-Fever & noisy breathing.Sepsis screen +. Platelet count-30000 Presented to us on DOL 27 Periorbital edema+,petechia +, pallor+,mottling + Gross ascites,Hepato splenomegaly +

68. Case 4 HLH suspected Bone marrow-Hemophagocytes Clinical exome- C ompound heterozygous mutation [c.658G>C (p.Gly220Arg) and c.1519G>T (p.Glu507Ter)] of PRF-1 gene Received IVIG and Dexamethasone Expired on DOL 11.

69. Report

70. Case 5 Term/LSCS/Bwt-2.3 kg/SGA History of one male sibling death at 1 year of age with similar history of prolonged fever spikes. Received birth vaccines. BCG scar Postnatal history : Baby was well till 21 days of life D eveloped high grade fever spikes with episodes of loose stools Multiple courses of oral and iv antibiotics Positive findings : Multifocal nodules in lower lung fields Diarrhoea and LRTI Sterile blood cultures Splenic abscess CMV positive Parvo virus positive UTI Meningitis and ventriculitis Huge hepatosplenomegaly Pancytopenia

71. Case 5 Bone marrow-Histiocytes+ DHR assay – Positive for CGD NBT-No reduction compared to controls

72. Take home message Primary immunodeficiency diseases are not uncommon, but often underdiagnosed in general clinical practice. PIDs lead to various combinations of recurrent infections, autoimmunity, lymphoproliferation, granulomatous disease, atopy, and malignancy. Laboratory work up should be guided by meticulous history and examination, after ruling out other secondary immunodeficiencies. A detailed family history always gives important diagnostic clues to an underlying PID.

73. References Nelson Textbook of Pediatrics,21 st Edition Avery’s Disease of the Newborn,11 th Edition Practical approach to diagnosis and management of PID-Rossouw TM et al,2018 Diagnostic Approach to Primary Immunodeficiency disorders-M Madkalkar et al,2013 Approach to PID,M Madkaikar , A Mishra, K Ghosh, Indian Pediatrics 2013 McCusker and Warrington Allergy, Asthma & Clinical Immunology 2011

74. THANK YOU

75. IMMUNITY INNATE/ NATURAL IMMUNITY ADAPTIVE/ AQUIRED IMMUNITY Anatomical & Physical barriers Phagocytic cells (Neutrophils, Macrophages) Natural killer cells (NK cells) Complement system Humoral immunity (B cell immunity) Cell mediated immunity (T cell immunity) Antibody mediated Extracellular pathogens -bacteria Intracellular pathogens- viruses, fungi Delayed allergic reactions Transplant rejection

76. Site of infection Possible cause Upper respiratory tract Antibody or complement deficiency Lower respiratory tract Antibody deficiency/ complement deficiency/ T cell deficiency/ phagocytic defect Skin, internal organs Phagocytic cell defect Blood or central nervous system (meninges) T cell defect/ Antibody /complement deficiency Gastrointestinal Antibody or T cell defect Type of organisms Capsulated organisms (streptococcus pneumoniae, H. influenza) T cell/ antibody defect/ complement defect Intracellular bacteria (mycobacteria, salmonella) T cell defect Meningococci Terminal complement defect Bryant PA, South M. Arch Dis Child Educ Pract Ed 2014;99:8–12 .

77. DISORDERS OF ANTIBODY PRODUCTION

78. X LINKED AGAMMAGLOBULINEMIA (BRUTON DISEASE)* Defect in normal differentiation of lymphocytes beyond pre B lymphocyte stage Defect in gene encoding cytoplasmic enzyme- Bruton tyrosine kinase Complete failure to produce immunoglobulins Cell mediated immunity normal Clinical features- rarely presents clinically in the neonatal period, KREC screen for neonatal diagnosis Recurrent pyogenic infections starting at 4-6 month of age 50% present before 1 year of age, 90% before 5 year of age Most common- sinopulmonary infections (otitis media, pneumonia) Others- gastroenteritis, pyoderma, septic arthritis, meningitis Minimal tonsillar tissue & no palpable lymph nodes on examination

79. Lab diagnosis of humoral defect SCREENING TEST CBC with differential count Immunoglobulin profile (quantitative Ig)- IgG, IgA, IgM, IgE Baseline & post immunisation titres- protein and polysaccharide antigens (e.g. pneumococcal, diphtheria, tetanus) ADVANCED TEST B cell maturation assay by flow cytometry B cell signalling assays KREC analysis Genetic mutation analysis (known) BTK, ATM mutations - Jordan K Abbot, Pia J Hauk, CPDT 2017

80. Complement defects

81. COMPLEMENT DEFECTS Rare- 1% population Severe disease- inherited as AR pattern with absolute deficiency of complement component Predisposition to autoimmune diseases – SLE (90% in deficiency of C1q) Recurrent pyogenic infections - streptococci, H. influenza (as opsonisation/ binding of antibody & complement critical for elimination) Most severe- C3 deficiency Deficiency of later components (C5-C9) – failure of membrane lysis Deficiency of C1 esterase – spontaneous episodes of local edema without urticaria (hereditary angioedema)

82. COMPLEMENT DEFECTS Ear infections, pneumonia, bacteremia, meningitis Angioedema, laryngeal edema, abdominal pain Laboratory tests Laboratory tests Complement screening assays: CH50, AH50 Specific assays: complement components C1 esterase inhibitor If Abnormal Treatment Treatment Prophylactic antibiotics, immunise with pneumococcal & N. meningitidis vaccines Monitor for autoimmune diseases Prophylaxis with androgens, infusion of C1esterase inhibitor for acute attacks

83. LABORATORY EVALUATION OF PHAGOCYTE DEFECT SCREENING TEST CBC with differential count (absolute neutrophil count- ANC) ADVANCED TEST Neutrophil oxidative burst assay -Dihydrorodamine assay (DHR) -Nitro blue tetrazolium test(NBT) Adhesion molecule assays (CD11B/CD18) Chemotaxis studies Mutation analysis

84. Defects in Cell Surface Protein Expression X-linked hyper IgM syndrome CD4+ T lymphocytes studied for the expression of CD40 ligand (CD154) SCID associated with a defect in the expression of the IL7R alpha chain and specific CD3 chains. LAD type 1 defect in β2 integrin expression can be diagnosed by evaluating for the presence of cell surface CD18 LAD type 2 defective fucosylation , failure of CD15s ( Sialyl Lewis X antigen) expression together with red blood cell typing that yields the rare Bombay blood type

85. Defective Intracellular Protein Expression Polyendocrinopathy , enteropathy , X-linked (IPEX) evaluating CD4+ T cells for surface expression of CD25 together with intracellular expression of FOXP3. In DOCK8 deficiency intracellular DOCK8 expression (absent or diminished levels of protein Familial hemophagocytic lymphohistiocytosis (FLH) Intracellular slam associated protein (SAP), x-linked inhibitor of apoptosis (XIAP) and perforin

86. Types of infections One site of infection > 2 sites of infection May be PID Generally not PID Physical examination Absent tonsils & lymph nodes Generally PID Recurrent otitis media, Sinopulmonary infections, Sepsis, meningitis , GI infections Autoimmune disorders Tonsils & lymph nodes present May be PID Laboratory evaluation EVALUATION OF SUSPECTED HUMORAL IMMUNE DEFECT CBC & Differential count Quantitative Immunoglobulins Specialised testing Treatment – Immunoglobulin replacement

87. Recurrent bacterial infections Serum immunoglobulin assays (Ig below 2SD for age) IgG, IgA or IgM B lymphocyte enumeration B cells absent B cells> 1% X linked- Bruton agammaglobulinemia AR- agammaglobulinemia μ heavy chain deficiency IgG IgA, IgM or N Common variable immunodeficiency (CVID) Hyper IgM syndrome Healthy infant, no increased bacterial infections, normalisation at 4-5 year of age (Transient hypergammaglobulinemia of infancy) AR- hyper IgM IgA IgG subclass levels 1,2,3 Specific antibody responses Selective IgA deficiency IgA with IgG subclass deficiency IgA with specific antibody deficiency

88. Pluripotent stem cell Common lymphoid progenitor Myeloid Erythroid Megakaryocyte lineages Double negative T cell Pre-TCR,CD3 Double positive T cell, TCR αβ CD3,CD4,CD8 Single positive T cell, TCR αβ CD3,CD4 or CD8 THYMUS Gamma-delta T cell TCR γδ ,CD3 Helper T cell TCR,CD3,CD4 Cytotoxic T cell TCR,CD3,CD8 NK cell CD16,CD56 Pre B cell Immature B cell BONE MARROW SPLEEN Transitional B cell Lymph node germinal centre Mature B cell CD19,CD20,IgM,IgD Memory B cell CD19, CD20 IgG, IgA, IgE Plasma cell Immunoglobulin LYMPHOCYTE DEVELOPMENT

89. Screening for PIDs Screening for SCID relies on polymerase chain reaction technology Based on process of germline gene rearrangements, which are unique to T and B cells Clusters of genes that encode variable (V), joining (J), and diversity (D) segments are spliced and recombined so that each individual cell has only 1 of each type of gene segment. During splicing, short TRECs are created, which are circular and contain a stable sequence that can be detected by primers. - Puck JM, J Clin Immunol 2012

90. Lab Evaluation of Suspected Cellular Immune defect INITIAL TESTS CBC with differential count (absolute lymphocyte count) Quantitative immunoglobulins Flow cytometry & Immunophenotyping -T, B, NK cell counts ADVANCED TESTS Based on initial test results or high clinical suspicion DTH (delayed type hypersensitivity testing) Lymphocyte proliferation to mitogens Cytotoxicity studies ADA or PNP levels of RBC TREC assay Genetic evaluation Advanced flow studies- TH17, CD40L, WASp etc.

91. COMBINED IMMUNODEFICIENCY(CID) Defects in both the T- and B-cell compartments, potential to cause the same diverse types of infections as SCID. Not the same risk of mortality in the first year after birth if untreated. Genetic overlap between SCID & CID, with some gene mutations causing either disorder because of differences in functional consequences of the mutations. Present in the neonatal period less commonly than SCID