APPROPRIATE

INVESTIGATION FOR PRIMARY

IMMUNODEFICIENCY IN -ELINDA 3HELLEY 3UCHARD, BSc, MB BCh, FCPath (SA) Clin, MMed (Clin), DTM&H Molecular Medicine and Haematology, Facullty of Health Sciences, University of the Witwatersrand and National Health Laboratory Service, Johannesburg, South Africa 3UVARNA"ULDEO, MB ChB, FCPath (SA) Clin, MMed (Clin), DTM&H Clinical Pathology Laboratory, National Health Laboratory Service, Mahatma Gandhi Memorial Hospital, Phoenix, South Africa #ATHYVAN2OOYEN, MB ChB, FCPath (Virol), MMed (Path) (Virol) Department of Serology and Immunology, National Reference Laboratory, AmPath Laboratories !"342!#4 Primary immunodeficiencies (PIDs) are rare but patients with recurrent infections are common. Every clinician needs an approach to appropriate laboratory investigation for confirming or excluding PID. Owing to the complexity of the immune system a staged diagnostic approach is utilised. The approach begins with excluding causes of secondary immune deficiency, followed by screening with a full blood count, white cell differential and antibody levels. Second-line tests include complement screens, antibodies to specific antigens and flow cytometry for T- and B-cell numbers. Third-line testing involves tests of cellular function, which are more specialised assays. These assays should be selected based on the presentation and history of the patient. The thirdline tests may lead to suggested genetic or molecular testing for confirmation of the diagnosis. Finally, all patients diagnosed with PID should be invited to participate in the South African Primary Immune Deficiency Registry. This will allow strengthening of diagnostic and therapeutic services for PID in South Africa.

).42/$5#4)/. Primary immunodeficiencies (PIDs) are a group of diseases caused by inherent defects of the immune system, which can present anywhere on a spectrum from severe and life-threatening, such as the paediatric emergency of severe combined immune deficiency (SCID), to mild recurrent infections presenting in teenage or adult patients. Although PID is rare, recurrent infections are common, making it necessary for every general practitioner to have a confident approach when investigating a patient with a suspected PID. The International Union of Immunological Societies expert committee1 lists eight categories of PID disorders: combined immunodeficiencies; well-defined syndromes with immunodeficiency; predominantly antibody deficiencies; diseases of immune dysregulation; congenital defects of phagocyte number, function or both; defects in innate immunity; autoinflammatory disorders and complement deficiencies.1

SOUTH AFRICA

For the purpose of laboratory work-up in a busy clinic however, it is still helpful to simplify these conditions into those that involve the innate immune system (including neutrophils, monocytes, complement), those of B cells and antibodies (humoral) and those of T cells (cellular). Despite new insights and molecular advances in the aetiologies of PID, the mainstay of diagnosis remains the patient’s history and family history. Understanding basic principles of immunology and applying these to the patient and family history and clinical examination, allows a focused approach to laboratory investigation for suspected PID.

02).#)0,%3 /& "!3)# )--5./,/'9 .%#%33!294/%6!,5!4%!0!4)%.47)4(0)$ The nature of the common infecting organisms can assist in identifying the possible associated immunodeficiency. Factors to note include whether the organisms are generally intracellular or extracellular organisms, whether they are encapsulated or non-encapsulated, and whether they are virulent or opportunistic. The human host attacks extracellular pathogens by sending protein mediators, such as antibodies and complement, and phagocytic cells to engulf the organism. For an intracellular pathogen however, complement and antibodies cannot reach the organism; therefore the host has no option but to kill the infected cell. Killing is accomplished by CD8+ cytotoxic T lymphocytes. Therefore, intracellular viral infections often elicit a CD8+ cytotoxic T-cell response, while extracellular organisms depend on antibody responses. Because B cells need help from CD4+ T lymphocytes in order to switch antibody isotype from IgM to IgG or other isotypes, CD4+ T cells are also important for an effective humoral immune response to extracellular organisms. This so-called ‘T-cell help’ refers to the interaction of CD40 on the B cell with CD40-ligand on the CD4+ T cell (Figure 1a and b), as well as appropriate cytokine production by the helper T cell. CD40 or CD40ligand deficiency causes a PID known as X-linked hyper-IgM syndrome, where the patient can produce copious IgM but cannot class-switch to IgG. It should also be borne in mind that as B cells are not very effective without T-cell help, any PID that causes T-cell dysfunction will secondarily result in B-cell dysfunction – so-called ‘combined’ immunodeficiency. The way the immune system ‘sees’ a protein antigen is the job of major histocompatibility complex (MHC), in humans known as human leucocyte antigen (HLA) molecules. Protein antigens are processed intracellularly to smaller peptide fragments. These fragments are presented on the surface of the cell via MHC molecules to responding T cells. Although intricacies have been described, in general, intracellular antigens are presented via MHC class I (corresponding to HLA-A, HLA-B and HLA-C) while extracellular antigens are taken up by professional antigen-presenting cells, processed to peptides inside an endosome, and presented on the surface of the cell via MHC class II (corresponding to HLA-DR, HLA-DP and HLA-DQ). Non-variant regions of MHC class I bind the CD8 molecule and MHC II binds

Correspondence: Melinda Suchard , e-mail [email protected]

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Current Allergy & Clinical Immunology, November 2012 Vol 25, No.4

to the CD4 molecule, allowing intracellular antigens to be presented to CD8+ T cells and extracellular antigens to CD4+ T cells. The T-cell receptor is unique to each T cell, which determines which CD4+ T cell or which CD8+ T cell can bind to the peptide within the MHCgroove (Figure 1a and b). As polysaccharide antigens are not displayed via MHC molecules, they cannot be ‘presented’ to CD4+ or CD8+ T lymphocytes. B cells may have receptors which bind polysaccharides; however B cells cannot present these polysaccharides to T cells and therefore cannot receive ‘T-cell help’ (Figure 1c). Antibodies to polysaccharides are therefore termed ‘T-independent’. While healthy neonates have functional T cells at birth, the T-independent response to polysaccharides (particularly production of IgG2) is not fully developed until after 2 years of age.2 We therefore vaccinate infants with protein or protein-conjugate vaccines, but do not

MHCI

Peptide FRAGMENT

expect infants to make robust antibody responses to polysaccharide vaccines until the age of 2 years.

53).'4(%0!4)%.4()34/294/$)2%#44(% ,!"/2!4/297/2+ 50 The decision to investigate should depend largely on the past medical history and physical examination. Warning signs include a history of severe, persistent, unususal (such as low virulence organisms or unusual sites of infection) or recurrent infections – abbreviated SPUR by the Immune Deficiency Foundation. Guidelines as to how many infections can be considered ‘unusual’ are available (Jeffrey Modell Foundation and European Society of Immune deficiency websites listed in Appendix A, p. 210) but patients need to be considered in the context of what is routinely seen in their local healthcare setting. The patient history alone can often

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Fig. 1. Processing and presentation of intracellular and extracellular antigens to the immune system. Modified from Milich et al.,3 Cooke et al.4 Janeway et al.5 and Somprayrac.6 Understanding the three pathways represented can assist the clinician to guide laboratory work-up of a patient with suspected immune deficiency towards the most likely pathway affected. Current Allergy & Clinical Immunology, November 2012 Vol 25, No.4

191

Table I. Important aspects to note in the patient history that can direct laboratory work-up towards the most likely avenue of primary immune deficiency !GEATONSETOFSYMPTOMS

Before or after 6 months of age? Relationship with time of weaning?

4YPESOFSYMPTOMSANDSIGNS - Abscesses - Presence/absence of pus - Non-healing lesions - Sterile site infections such as meningitis/septicaemia &REQUENCYOFSYMPTOMS

Is the frequency higher than that expected for that age group in that particlar healthcare setting?

3EVERITYOFINFECTIONS

Ask about previous hospitalisation or need for intravenous antibiotics – objective signs of severity Any previous sterile site infection?

4YPEOFORGANISMSCULTURED

Viral, bacterial, parasitic? Any unusual organisms? Note sensitivity results

&AMILYHISTORYPARTICULARLYIMPORTANT

Ask specific questions, e.g. did anyone in your family lose a child in infancy?

3ITESOFINFECTION

Superficial or deep? Sterile sites versus sites with commensal organisms? Typical or unusual sites for this organism?

!SSOCIATEDSKINRASHESORECZEMA !SSOCIATEDBOWELSYMPTOMS !SSOCIATEDAUTOIMMUNITYORFAMILY HISTORYOFAUTOIMMUNITY Adapted from Slatter & Gennery2 and Johnston.7

guide investigation of PID and all previous medical records with laboratory results must be reviewed. The age of presentation and organisms responsible for recurrent infections provide important clues (Table I).2,7 In general the more severe the immune defect, the earlier the presentation. Deficiencies of T lymphocytes tend to manifest within the first few months of life and result in the most severe pictures because of secondary B-cell dysfunction (‘combined immune deficiency’). Milder forms may present at older ages. Antibody disorders present after 6 months with declining maternal antibodies. Milder forms such as selective IgA deficiency, or specific antibody deficiencies (e.g. to particular antigens only, e.g. Streptococcus pneumoniae, Haemophilus influenzae) may present in early adulthood.2 Deficiencies of the innate immune system can manifest any time from birth, although may only manifest later in childhood. Complement disorders often manifest in adolescence or adulthood.2 Although there are overlaps, the nature of the infecting organisms can sometimes guide as to which avenue of immune deficiency is more likely. Phagocyte disorders result in infections with bacteria such as staphylococci, Serratia marcescens, Burkholderiacepacia, Klebsiella species, Escherichia coli, Salmonella species and Proteus species and fungi including Candida, Aspergillus and Nocardia species. Complement deficiencies often result in infection with pyogenic bacteria such as streptococci and H. influenzae (often together with autoimmunity), with deficiencies of late complement components predisposing to infections with Neisseria gonorrhoea and N. meningitides.8 B-cell and antibody deficiencies are often marked by predisposition to infection with pyogenic bacteria, enteroviruses (such as echovirus, poliovirus) and mycoplasma. T-cell disorders show a spectrum of pathogens very similar to those seen in HIV infection, including viruses like

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cytomegalovirus (CMV), adenovirus, molluscum; fungi such as Candida and Pneumocystis jiroveci, protozoa such as Cryptosporidium parvum, as well as pyogenic bacteria8 (Table II).

!002/!#( 4/ !002/02)!4% ,!"/2!4/29 ).6%34)'!4)/. First-line investigations which are used as screening tests Before pursuing a diagnosis of PID, more common causes of secondary immune deficiency or failure to thrive should first be excluded. Consider testing for HIV, tuberculosis, cystic fibrosis, coeliac disease, diabetes mellitus and protein-losing states where appropriate. For possible primary immune defects, first-line testing includes a full blood count with platelet count and white cell differential count, as well as immunoglobulin levels. In the white cell differential, particular regard should be paid to absolute numbers of the white cell subsets, rather than percentages, as with a low total lymphocyte count, the percentages can be normal despite low absolute numbers. Circulating lymphocytes comprise mainly T lymphocytes, which can be used as a screening test for T lymphopenia. Immunoglobulin deficiencies are responsible for most PIDs. Age-appropriate reference intervals must be used as immunoglobulins increase with age. Immunoglobulin concentrations in infants may be falsely elevated with maternal antibodies that are transferred across the placenta (IgG) or via breast milk (IgA). It is important to assess IgG, IgA, IgM and IgE together. IgG subclass evaluation (IgG1, IgG2, IgG3, IgG4) should not form part of the screening panel as it is a relatively expensive assay.

Current Allergy & Clinical Immunology, November 2012 Vol 25, No.4

Table II. Symptoms which may alert the clinican to an underlying primary immune deficiency (these are simplified examples and many other conditions would be included in the differential diagnosis) Symptoms

Possible underlying primary immune deficiency

Liver abscess in a child or recurrent superficial abscesses Fungal pneumonia

Chronic granulomatous disease

Delayed shedding of primary teeth Eczema Staphylococcal infections Aspergilloma Bone fractures Joint hyperextensibility Eosinophilia Candidiasis

Hyper IgE syndrome

Delayed separation of umbilical cord (>30 days) Peri-anal abscesses in neonatal period Absent or less pus than expected for lesions, poor wound healing

Leucocyte adhesion deficiency type 1 (Slatter & Gennery2) Neutropenia

Recurrent or unusual site of Neisserial infection

Deficiency of terminal complement components

Disseminated BCG Deep-seated non-tuberculous mycobacteria Salmonella species

SCID Mendelian susceptibility to mycobacterial disease (interleukin-12, interleukin-23, inteferon γ pathway)

Pneumocystis jiroveci pneumonia Recurrent bronchiolitis Recurrent candidiasis Cytomegalovirus disease Cryptosporidium parvum diarrhoea Molluscum

T-cell disorder, e.g. SCID or CD40-ligand deficiency

Recurrent pneumonia Arthritides Bronchiectasis Enteroviral meningoencephalitis (e.g. polio, Coxsackie virus, echovirus)

Agammaglobulinaemia

Recurrent upper respiratory tract infections, gastrointestinal disturbances Encapsulated organisms (such as Streptococcus pneumoniae, Haemophilus influenzae type B, Moraxella catarrhalis)

Selective IgA deficiency Common variable immune deficiency Specific antibody deficiency

Hypocalcaemia Absent thymic shadow Cardiac defects

DiGeorge syndrome

Erythroderma

Omenn syndrome

Hepatosplenomegaly Massive lymphadenopathy in infancy

(variant of SCID)

Midline ulceration

MHC class I deficiency

Recurrent angio-oedema

C1 esterase deficiency

Eczema Thrombocytopenia Recurrent infections

Wiskott-Aldrich syndrome

NFκB pathway

Adapted from Slatter & Gennery,2 Johnston,7 Oliveira and Fleisher8 and De Vries.9 SCID – severe combined immune deficiency; MHC - major histocompatibility complex.

Second-line investigations In second-line investigations B lymphocytes, T lymphocytes and natural killer cells are enumerated by flow cytometry, which uses fluorescent labelled monoclonal antibodies to label specific markers on cells. An absence of B lymphocytes is noted in agammaglobulinaemia and absence of T cells in severe combined immune deficiency (SCID). Natural killer cells and B lymphocytes can also be reduced in SCID.

Antibody function can be assessed by evaluating responses to specific vaccines. Protein and polysaccharide vaccine responses should be measured, but vaccine responses to polysaccharide antigens are unreliable in children below the age of 2 years. Haemolytic assays are used to identify deficiencies in the complement cascade. If a deficiency is noted tests to measure some specific components are available.

Current Allergy & Clinical Immunology, November 2012 Vol 25, No.4

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Third-line investigations to measure function T-cell function can be measured by performing in vitro assays to assess T lymphocyte proliferation to specific antigens or non-specific mitogens. Additionally, antigen-specific cytokine responses can be measured. Additional phenotyping of T cells can also be performed, including the enumeration of naïve and memory T cells, determining TCR type (αβ /γδ) and HLA-DR expression on T cells. Antibody responses to specific antigens can be measured by vaccinating the patient (with non-live vaccines) and measuring pre and post (4 weeks later) immunisation antibody titres. A twofold increase in antibody titres should be seen with polysaccharide vaccines and a fourfold increase with protein vaccines. Immunoglobulin subclasses (IgG1, IgG2, IgG3 and IgG4) can be used in selected patients to delineate subclass deficiencies if IgG levels are normal but antibody responses are defective. Subclass deficiencies are also seen in patients with IgA deficiency. Class-switched memory B cells can be determined in patients with common variable immunodeficiency (CVID) for further subclassification and as a predictor of outcome and complications. Tests are available to evaluate the ability of neutrophils to migrate to the site of infection, phagocytose the pathogen and kill by oxidative methods. The inablilty to migrate is seen in leucocyte adhesion defects, while the lack of oxidative killing is indicative of chronic granulomatous disease. In addition to haemolytic assays to determine functionality of the classic and alternative complement pathways, assays are available to evaluate the mannosebinding lectin (MBL) complement pathway. Patients with defects in this pathway may be asymptomatic, but may present with recurrent pyogenic infections, as well as susceptibility to mycobacterial, fungal or atypical bacterial infections. Natural killer cell function can also be investigated in patients with normal natural killer cell absolute counts who suffer from recurrent herpes virus infections.

Fourth-line investigations to confirm the diagnosis Access to highly specialised stage 4 investigations are limited in South Africa. Some of the available tests include Bruton’s tyrosine kinase assays to confirm agammaglobulinaemia, CD40-ligand for hyper-IgM syndrome, CD 11/18 determination for leucocyte adhesion deficiency and common gamma chain detection for X-linked SCID. Confirmation of the molecular defect is not, however, necessary to commence therapy or to include the patient on the PID registry.

02)-!29)--5.%$%&)#)%.#92%')3429 Any patient with a diagnosed PID should be invited to be included on the South African Primary Immune Deficiency Registry. This national registry is coordinated through the NHLS laboratory at Tygerberg Hospital, but includes patients from throughout South Africa, in both state and private sectors. The registry is essential to improve our understanding of the spectrum of PIDs seen and to allow estimates of prevalence of these disorders. It becomes a vital tool for advocacy for improved diagnostics and treatment. It may also allow identification of diseases more common in South Africa than elsewhere. Patients may benefit from notification if new testing or treatment for their disease becomes available in South Africa. The consent forms and

194

notification forms can be requested from the laboratory contact numbers in Appendix A, p. 210). The five tiers of testing are summarised in Table III.

5.$%234!.$).' 3!-0,% 2%15)2%-%.43 &/2)--5./,/')#!,!33!93 Sample requirements for the laboratory assays can be generalised as follows. Any tests for protein components, such as complement or immunoglobulins, require a small sample volume (2-3 ml) of clotted blood and can reach the specialised laboratory within 4872 hours of blood draw. Flow cytometry for absolute numbers of cells, such as T or B cells, requires a small volume (2-3 ml) of anticoagulated blood (EDTA or heparin, depending on the laboratory) which can reach the laboratory up to 48-72 hours after blood draw. For tests of cellular function and metabolism where larger blood volumes are required (5-10 ml), blood must reach the laboratory within 6 hours of blood draw so that the cells are still viable. For functional assays, laboratories may require the clinician to send blood from a healthy age-matched control (allowing the laboratory to control for factors such as transport time, transport temperature, age of patient). Functional assays include lymphocyte proliferation assays and tests for oxidative burst or phagocytosis. These assays are best booked with the laboratory in advance. A point to consider is that acute infection or inflammation can alter many of the lymphocyte subsets and immunoglobulin levels, so ideally tests for PID should be performed when the patient is relatively healthy. If this is not possible and tests are done during periods of illness, the assays should be repeated when the patient is healthy again. The patient’s age must be reported accurately to the laboratory to allow use of age-appropriate reference intervals.10,11 It should also be borne in mind that children are not expected to have fully developed T-independent responses, e.g. to polysaccharide antigens, for the first 2 years of life.2

,/')34)#!, $)&&)#5,4)%3 7)4( 3!-0,% 4%34).' South Africa is fortunate to have many laboratories able to perform screening assays and immunoglobulin assays. However, the specialised tests of lymphocyte function are still not widely available, and can only be performed at certain tertiary institutions or private laboratories; this limits accessibility as functional assays require blood to reach the laboratory within 6 hours of blood draw.

-!.!'%-%.4/&4(%0!4)%.4$52).'4(% ,!"/2!4/297/2+ 50 Prompt diagnosis of PID is important for the peace of mind of the patient, as well as for selection of appropriate therapies. Specific guidelines should be consulted for each condition, but a few general points can be borne in mind. For severe antibody deficiencies, intravenous or subcutaneous immunoglobulin has revolutionised quality and length of life of patients. Replacement of immunoglobulins can prevent frequent hospitalisations and chronic sequelae such as bronchiectasis.12 For severe T-cell disorders, haemopoietic stem cell transplant from an HLA-matched sibling may cure the condition. For many conditions in which immunoglobulin replacement or transplant is not indicated, supportive measures such as prophylactic antibiotics or antifungals can be used with

Current Allergy & Clinical Immunology, November 2012 Vol 25, No.4

Table III. Laboratory investigations should follow a tiered approach and be directed towards the most likely avenue of immune deficiency, as directed by the patient history, family history and clinical examination First-line screening tests ,ABORATORYTEST Full blood count, platelet count, differential count

.OTES Pay attention to absolute numbers of white cell subsets in the differential, rather than percentages The total lymphocyte count may be helpful in babies born to families with a history of previous infant death from unknown causes (does not definitively exclude severe combined immune deficiency, so in families with a history of combined immune deficiency, flow cytometry is needed)

Immunoglobulin classes (IgG, IgM, IgA and IgE)

IgE often needs to be requested separately. IgE should be tested in patients who may be at risk of hyper IgE syndrome

Exclude causes of secondary immunodeficiency, e.g. HIV test, TB testing, etc.

Depends on patient presentation and examination

Second-line testing for cellular, humoral or innate deficiency – depends on suggestive history, symptoms and cultured organisms Cellular

Humoral

Innate

Laboratory test

Notes

Laboratory test

Notes

Laboratory test

Notes

T-cell and natural killer cell numbers by flow cytometry

(usually includes B-cell numbers)

Immunoglobulins to specific antigens, e.g. Streptococcus pneumonia, Candida, tetanus, Haemophilus influenzae

Select a protein and a polysaccharide antigen Can select an antigen with which the patient suffers recurrent infections, e.g. Candida, to look for a specific antibody defect

Total classic complement (CH50 or CH100)

Send on ice

B cell numbers by flow cytometry

Usually included with T and natural killer cell numbers

Total alternative complement (AH50 or AH100)

Send on ice

Third-line testing – tests for function Cellular Lymphocyte proliferation assay to mitogens

Requires viable cells – must reach laboratory within 6 hours

Lymphocyte proliferation assay to specific antigens

Requires viable cells – must reach laboratory within 6 hours Antigens can be selected according to patient history e.g. Candida for recurrent candidal infections etc. Requires viable cells – must reach laboratory within 6 hours For suspected SCID Does not require viable cells

T cell cytokine production to mitogens and antigens Naïve/memory T cells

T-cell receptor type (αβ / γδ)

Humoral Vaccinate patient* and re-test the specific antibodies, e.g. to tetanus, H. influenzae, S. pneumoniae Immunoglobulin subclasses, e.g. IgG1, IgG2, IgG3, IgG4

Do not administer live vaccines

Class-switched memory B cells

Useful for common variable immune deficiency

Does not require viable cells

Current Allergy & Clinical Immunology, November 2012 Vol 25, No.4

Innate Oxidative burst test

Requires viable cells – must reach laboratory within 6 hours

Chemotaxis testing

Requires viable cells – must reach laboratory within 6 hours

Phagocytosis testing

Requires viable cells – must reach laboratory within 6 hours

Specific complement components, e.g. C3, C4 Mannose binding lectin complement pathway

Send on ice

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Table III (continued) Fourth-line testing – genetic testing and specialised immunological assays CD40 and CD40-ligand testing

If X-linked hyper IgM syndromes suspected

CD11 and CD18 on granulocytes for leucocyte adhesion deficiency

Often included in second-line flow cytometry for T- and B-cell numbers

Common gamma chain testing

If X-linked SCID is suspected

BTK testing

If X-linked agammaglobulinaemia is suspected

Tests for specific genetic defects

With suggestions from laboratory

Fifth-line – Primary Immune Deficiency Registry Invite patient to register on Primary Immune Deficiency

Does not require a specific molecular diagnosis

Registry - Sign informed consent - Fill history and laboratory information questionnaire *Do not administer live vaccines to a patient suspected of immune deficiency. SCID – severe combined immune deficiency; BTK – Bruton’s tyrosine kinase.

great success. Antibiotic choices include trimethoprim sulfamethoxazole which can be used for B- and T-cell defects and the antifungal itraconazole which is recommended for patients with chronic granulomatous disease. Azithromycin is a good second-line choice for patients with humoral immunodeficiencies. More specific therapies are available for selected deficiencies, such as interferon-gamma replacement for patients with chronic granulomatous disease. Severe T-cell PID should be considered a paediatric emergency.9 As discussed, T-cell deficiencies cause secondary B-cell dysfunction, giving the patient a combined immune deficiency syndrome. Patients with T-cell deficiencies therefore require isolation, strong infection control measures and urgent referral in severe cases for possible haemopoietic stem cell transplant. Patients awaiting transplant may need to be managed by the primary physician or paediatrician until such time as all tissue typing on possible HLA-matched siblings has been completed. Tissue typing of patient and siblings should be started as soon as possible. Stringent infection control measures should be followed during the work-up, including irradiation and leucodepletion of blood products, to prevent graft-versus-host disease or infective risk. Live vaccines such as Bacille Calmette Guérin (BCG), oral polio vaccine and varicella vaccination are strictly contraindicated.

Disseminated BCG infection can therefore pose barriers to bone marrow transplantation. In cases where a baby is born to a family with a history of SCID, the utmost attention should be paid to avoiding BCG vaccination. This includes not sending the baby to the nursery and fully educating family and nursing staff in the ward. Prenatal genetic testing and counselling can prepare the family. For pregnant mothers who have previously lost an infant to infectious causes, consider performing a full blood count with differential for total lymphocyte count on the infant prior to BCG vaccination. Most patients with SCID are lymphopenic, although a normal lymphocyte count does not exclude SCID.2 Flow cytometry for T-cell numbers is needed in high-risk cases.

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Vaccination with live vaccines must be avoided in patients with T-cell immune deficiencies or severe humoral deficiencies. Live vaccines include oral polio, BCG, measles, mumps, rubella, varicella, rotavirus and yellow fever. Additionally, live vaccines should be used with caution in the close relatives of patients with severe immune deficiency, such as siblings, as they may be able to transmit the vaccine strain to the patient. Nonlive vaccines assume even greater importance however and should be given to both the patient and the family members. Thorough vaccination of the immediate family can protect the patient from acquiring infections. Therefore in siblings of diagnosed patients, inactivated polio vaccine should be given instead of live oral polio vaccine. In times of outbreaks, hyperimmune globulins can be actively sought and used where necessary for patient and siblings, e.g. varicella hyperimmune globulin during a chickenpox outbreak. As BCG vaccination in South Africa is given at birth, patients with severe immune deficiencies have often already been vaccinated at time of diagnosis.

Dr Melinda Shelley Suchard and Dr Suvarna Buldeo are employed by the National Health Laboratory Service. Dr Cathy van Rooyen is employed by AmPath Laboratories. These laboratories offer diagnostic testing for primary immune deficiency. The authors have no other potential conflict of interests.

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#/.#,53)/. Laboratory testing is available in South Africa for many of the more common PIDs. A stepwise approach to laboratory testing should be followed, allowing confirmation or exclusion of PID in most cases. The patient and family histories remain the cornerstone of diagnosis. Even in cases where the molecular defect cannot be identified, a PID may be strongly suspected on the basis of history alone. Growing the PID registry may assist in building capacity for improved diagnostics and therapeutics for PID in South Africa.

2%&%2%.#%3 1. Al-Herz W, Bousfiha A, Casanova JL, et al. Primary immunodeficiency diseases: an update on the classification from the International Union of Immunological Societies expert committee for primary immunodeficiency. Front Immunol 2011;2:54. 2. Slatter MA, Gennery AR. Clinical immunology review series: an approach to the patient with recurrent infections in childhood. Clin Exp Immunol 2008;152(3):389-396. 3. Milich DR, Chen M, Schodel F, Peterson DL, Jones JE, Hughes JL. Role of B cells in antigen presentation of the hepatitis B core. Proc Natl Acad Sci U S A 1997;94(26):14648-14653. 4. Cooke MP, Heath AW, Shokat KM, et al. Immunoglobulin signal transduction guides the specificity of B cell-T cell interactions and is blocked in tolerant self-reactive B cells. J Exp Med 1994;179(2):425438. 5. Janeway CA, Travers P, Walport M, Shlomchik MJ. The humoral immune response. Chapter 9. Immunobiology. New York: Garland Science, 2005.

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6. Sompayrac L. How the Immune System Works, 4th ed. Oxford: Wiley-Blackwell, 2012:28,46,55.

AIDS Clinical Trials Group P1009 study. J Allergy Clin Immunol 2003;112(5):973-980.

7. Johnston SL. Clinical immunology review series: an approach to the patient with recurrent superficial abscesses. Clin Exp Immunol 2008152(3):397-405.

9. Comans-Bitter WM, de Groot R, van den Beemd R, et al. Immunophenotyping of blood lymphocytes in childhood. Reference values for lymphocyte subpopulations. J Pediatr 1997;130(3):388-393.

8. Oliveira JB, Fleisher TA. Laboratory evaluation of primary immunodeficiencies. J Allergy Clin Immunol 2010;125(2 Suppl 2):S297-305.

10. Rose ME, Lang DM. Evaluating and managing hypogammaglobulinemia. Cleve Clin J Med 2006;73(2):133-137,140,143-144.

9. De Vries E. Patient-centred screening for primary immunodeficiency, a multi-stage diagnostic protocol designed for non-immunologists: 2011 update. Clin Exp Immunol 2012;167(1):108-119.

&524(%22%!$).' McPherson RA, Pincus MR. Immunodeficiency disorders. Chapter 50. Henry’s Clinical Diagnosis and Management by Laboratory Methods. 22nd ed., Philadelphia: Elsevier, 2011: 963-973.

8. Shearer WT, Rosenblatt HM, Gelman RS, et al. Lymphocyte subsets in healthy children from birth through 18 years of age: the Pediatric

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