Humoral

Complement is the major humoral component of the innate response. When the central component, C3b, which can be activated by at least three pathways, is bound to an antigen, it interacts powerfully with C3b receptors on neutrophils and monocytes, enhancing phagocytosis. C3b also initiates a cascade activating further components such as C5–C9, which lyse cell membranes, a mechanism particularly important in the handling of systemic neisserial infections.

Mannan-binding lectin, which binds to mannose on the surface of microorganisms, activates the classical complement pathway; this is important in the early stages of infection before antibody production, particularly in young children whose antibody-producing ability is immature.

Alpha- and beta-interferon (IFN-α, IFN-β) proteins are produced by virally infected cells. They render other cells immune to virus infection by producing an antiviral state. They also increase natural killer (NK) cell activity and increase human leukocyte antigen (HLA) class I antigen expression on cells. Gamma-interferon (IFN-γ), though having some antiviral effect, upregulates immune responses by enhancing intracellular killing of microorganisms such as mycobacteria.

Many bacteria require iron for growth, and decreasing iron availability is one mechanism of defence used by the host. An avid iron-binding protein, lactoferrin, present in human milk, reduces the growth of Escherichia coli . The removal of serum iron, which occurs during infections, increases the bacteriostatic effect of serum.

Lysozyme is found in neutrophil lysosomes and body secretions, including tears, saliva, and at increased concentrations on newborn skin, and has antibacterial properties.

Cellular

Phagocytic cells such as neutrophils and monocyte/macrophages migrate to the site of infection attracted by products of the acute inflammatory response (chemotaxis), including cleaved complement fragments. Adherence to the bacterium and ingestion (phagocytosis) follow, aided by immunoglobulin G (IgG) and complement (C3b), which stick to bacteria and phagocyte cell surface receptors Fcg and CR1/3, respectively (opsonisation). Following phagocytosis, lysosomes fuse with the bacterium-containing phagosome and release lysosomal enzymes (e.g. myeloperoxidase) and free oxygen radicals, which kill and digest microorganisms. Neutrophil numbers and function are enhanced by the inflammatory response.

Monocytes and macrophages kill extracellular bacteria more slowly and less efficiently than neutrophils but are important in defences against intracellular microorganisms, such as Mycobacterium tuberculosis and Listeria monocytogenes , because their activity is greatly enhanced by cytokines, particularly IFN-γ, produced by T lymphocytes.

NK cells are part of the non-T, non-B-cell lymphoid population and recognise and kill tumour and virus-infected cells.

Antibodies

These are produced by B lymphocytes and their derivatives, plasma cells, in response to specific antigens such as microbial or vaccine peptide.

The immunoglobulin class (isotype) is determined by the heavy chain. During B-lymphocyte development, genes coding for the variable and constant parts of the immunoglobulin chain are rearranged. One each of the variable (V), diversity (D) and joining (J) heavy-chain genes are rearranged to lie adjacent to the relevant heavy-chain constant-region gene, so that transcription produces a single protein with a constant and a variable portion ( Fig. 39.2 ). Approximately 10 ¹⁶ different specific B-lymphocyte receptors (with a similar number of TCR) are generated. Further rearrangement to bring a particular variable gene combination to lie adjacent to a different heavy-chain constant-region gene allows the B lymphocyte to switch to another antibody isotype with the same antigen specificity. The m chain is always produced first; class switching to other isotypes occurs later. Specific antibody diversity is created by the large number of possible combinations of variable-region genes that can be selected and is increased further by a very high mutation rate in V genes. A similar but slightly less complex process takes place with light chains, of which there are two types, kappa and lambda.

Immunoglobulin heavy- and light-chain gene rearrangements. Discrete segments of the V, D and J heavy-chain genes are rearranged to lie adjacent to the relevant heavy-chain constant-region gene. Light-chain gene rearrangements using V and J segments from either kappa or lambda light chains complete the process. Transcription produces a single protein with a constant and a variable portion.

(Adapted with permission from .)

IgM is the first antibody class produced in primary immune responses. For most antigens, there is a subsequent switching to other classes. Its large pentameric structure confines it to the intravascular space, where it fixes complement.

IgG is the main class of antibody produced in secondary immune responses. Functions include opsonisation, complement fixation leading to C3 opsonisation and complement-mediated lysis, neutralisation of toxins or viruses, and participation in antibody-dependent cellular cytotoxicity. Though there is considerable overlap, the four subclasses of IgG tend to have different functions. Responses to protein antigens are mainly in the IgG _l and IgG ₃ subclasses. IgG ₂ binds polysaccharide, such as group B streptococcal surface antigen. The role of IgG ₄ is, as yet, unclear.

IgA is the main antibody in secretions, where it forms a dimeric molecule. In addition to protecting mucous membranes and the gastrointestinal tract from infection, IgA may have a role in limiting food antigen uptake from the gut. Serum IgA is mainly in monomeric form, though dimeric and trimeric forms are also found. It is secreted in breast milk.

Mast cells and basophils have receptors for the Fc part of IgE. Antigen binding causes degranulation of the cells, triggering an immediate hypersensitivity response.

The final stages of B-lymphocyte differentiation require stimulation with the appropriate antigen, leading to maturation into plasma cells. The full process requires the cooperation of antigen-specific T helper (CD4+) lymphocytes. Isotype switching from IgM production to the other antibody classes is critically dependent on T/B-lymphocyte interaction via the B-lymphocyte CD40 antigen and the CD40 ligand expressed on activated T lymphocytes (CD40L, CD154).

Cell-mediated immunity

T (thymus-derived) lymphocytes kill intracellular microbes and regulate immune responses by secreting cytokines and growth factors.

T lymphocytes develop from bone marrow stem cells, but mature in the thymus, where their receptor genes (TCR) are rearranged to generate the diversity of lymphocyte receptors needed to counter every potential antigen. Self-reactive T lymphocytes are deleted and only those recognising antigen in association with self MHC are preserved and exit the thymus. Regulatory CD4+ T lymphocytes (T _REG ) help maintain peripheral tolerance to self antigens.

The TCR is similar to the immunoglobulin molecule, with constant and variable parts and the generation of diverse specificity achieved in a similar way. Recombinase activating genes 1 and 2 ( RAG1, 2 ) are critical for TCR gene rearrangement. During thymic maturation, T lymphocytes also acquire important functional surface molecules. CD3 is closely associated with the TCR. The CD4+ T-helper lymphocyte is the primary effector cell. Once switched on by antigen presentation via the MHC II molecule, these cells develop either by the T _H 1 route, with production of the cytokines interleukin-2 (IL-2) and IFN-γ, stimulating macrophage function, cell-mediated immunity and B-lymphocyte class switching to IgG ₂ production, or to T _H 2 cells, which produce predominantly IL-4 and IL-10, which promote antibody responses and class switching, particularly towards IgG ₁ , IgG ₄ and IgE. Allergic responses and those against parasites are of T _H 2 type. Antigen responses may follow a T _H 1 or T _H 2 route, depending on a complex set of circumstances. CD8+ cytotoxic lymphocytes are activated by antigen presentation via the MHC I molecule, and directly kill virally infected cells.

Haematology

A full blood count and blood film examination will readily detect neutropenia. Bone marrow aspiration will distinguish between failure of production and increased peripheral destruction, and exclude a myelodysplastic or malignant process. Neutrophilia in the absence of overt infection may suggest a neutrophil adhesion defect or functional problem (e.g. CGD). Lymphopenia, using appropriate age-related ranges, is highly suggestive of a combined immune deficiency of primary or secondary aetiology ( ), but a normal lymphocyte count does not exclude SCID. A manual differential will differentiate nucleated red cells in infants and abnormal leukocyte morphology in sick children. Abnormal leukocyte granules are characteristic of Chédiak–Higashi syndrome. Platelet volume is universally low in Wiskott–Aldrich syndrome (WAS). Eosinophilia may be marked in Omenn syndrome, immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome and hyper-IgE syndrome.

Tests of innate immunity

Complement function should be assayed by testing the ability of patient serum to lyse sensitised red blood cells, thus testing the whole pathway. Deficiency in any one component will result in a failure of lysis. Samples must be separated and frozen within 2 hours of venesection. If repeat testing shows a persistent abnormality, evaluation of individual complement components should be performed. Mannan-binding lectin deficiency is detected by analysing the genotype; complete deficiency may be associated with severe infection.

Neutrophil function tests are technically difficult, as neutrophils activate and then quickly die after venesection. The nitroblue tetrazolium (NBT) test is a rapid and sensitive test for CGD, but false normal results can be seen when the test is performed infrequently; it is now being superseded by the more reliable flow cytometric evaluation of the neutrophil oxidative burst.

Adaptive immune system

Definition of molecular defects

The genetic basis of an increasing number of immune deficiencies is well defined. Usually, a gene defect coding for the protein results in no protein expression, expression of low amounts or expression of abnormally sized protein, and can be detected by Western blotting and flow cytometry ( ). In the presence of an appropriate history or abnormal protein expression, genetic analysis may be undertaken.

General features of severe combined immunodeficiency

Affected infants appear well at birth but within the first few months of life fail to clear infection and present with persistent respiratory tract or gut infection, failure to thrive and, sometimes, apparent food intolerance ( ). Persistent respiratory tract infection with respiratory syncytial virus or parainfluenza viruses is common, with failure to clear virus accompanying persistent bronchiolitic-like signs. An insidiously progressive persistent respiratory infection with radiological evidence of interstitial pneumonitis should raise the suspicion of Pneumocystis jiroveci infection, often a co-pathogen with respiratory viruses ( ). Other presentations include prolonged otitis media and invasive bacterial infections, particularly staphylococcal or Pseudomonas septicaemia and pneumonia, which may respond poorly to appropriate treatment. Severe invasive fungal infection is rare, but often fatal. Extensive persistent superficial candidiasis is more common. Occasionally infants present with disseminated bacille Calmette–Guérin (BCG).

Congenital graft-versus-host disease (GvHD) occurs because many infants with SCID are unable to reject foreign lymphocytes, acquired either from the mother in utero ( ) or from a non-irradiated blood transfusion. When the disease is clinically apparent, there is typically a mild reticular skin rash, sometimes with abnormal liver function tests. GvHD following transfusion with non-irradiated blood or white cell or platelet concentrates is generally more severe and can be fatal. In these cases, the skin rash is severe and lymphadenopathy and hepatosplenomegaly may be present. The clinical picture may be indistinguishable from Omenn syndrome, but identification of maternal cells by karyotype or DNA fingerprinting will distinguish maternofetal GvHD from Omenn syndrome.

Examination usually reveals a wasted child who has dropped through the growth centiles, with evidence of candidiasis and other infections. Skin rashes may indicate infection or GvHD. There is no clinically detectable lymphoid tissue. There may be hepatomegaly.

Investigation usually shows severe lymphopenia from birth. It should be remembered that in infancy a normal lymphocyte count is >2.7 × 10 ⁹ /l (unlike adults, where it is >1.0 × 10 ⁹ /l). Lymphocytic phenotyping shows severely depleted T-lymphocyte numbers; B lymphocytes and NK cells may be present or absent, depending on the type of SCID. Occasional variants show unusual patterns of immature T-lymphocyte markers; in such cases, maternal engraftment should be excluded. Mitogen responses are usually absent. Immunoglobulin estimations show low levels of IgG, IgA and IgM but residual maternal IgG may give a falsely reassuring result and it can be difficult distinguishing IgA and IgM levels in SCID from the low levels seen in normal infants. Isohaemagglutinins are a useful measure of in vitro IgM production, and absence is significant. If SCID is suspected, lymphocyte phenotyping is more reliable than immunoglobulin estimation. Chest radiographs show an absent thymus and, if infection is present, hyperinflation and/or interstitial pneumonia.

In children who die, postmortem examination reveals severely depleted lymphoid tissue, with nodes and thymus showing no lymphoid follicles and absent Hassall’s corpuscles in the thymus. Without treatment, patients die from infection by about 12 months of age. Currently, the only curative treatment is haematopoietic stem cell transplantation (HSCT), although clinical gene therapy trials for common gamma chain and ADA deficiency are in progress. Supportive interim treatments include antibiotic prophylaxis with co-trimoxazole as anti- Pneumocystis treatment, antifungal prophylaxis and immunoglobulin replacement. Live vaccines, including BCG, must be avoided. The diagnosis of SCID is a paediatric emergency, and suspected cases should be urgently referred to a designated treatment centre for assessment and treatment.

Newborns suspected of having a severe immunodeficiency disorder should be protected using isolation techniques, including limitation of the numbers of persons involved with care; individuals with respiratory or gastrointestinal symptoms of infection should avoid contact. If the mother is CMV-negative, breastfeeding should be encouraged; otherwise it should be discontinued to prevent neonatal CMV infection being transmitted through the milk. Wherever the child is managed, the importance of strict hand-washing procedures cannot be overemphasised. Blood products should be CMV-negative and irradiated to avoid the risk of transfusion GvHD.

Types of severe combined and combined immunodeficiencies

Omenn syndrome

Omenn syndrome is characterised by a generalised erythematous rash, together with scaling and erythroderma, lymphadenopathy, hepatosplenomegaly, increased serum IgE levels with a marked eosinophilia and the usual clinical features of SCID ( Fig. 39.6 ) ( ). The syndrome is a ‘leaky’ form of SCID in that small numbers of very abnormal T lymphocytes ‘leak’ past the block in T-lymphocyte development. The underlying defect in many cases is a mutation in the RAG genes, although other gene mutations have been associated with the phenotype ( ). HSCT is the only curative treatment.

Omenn syndrome. A newborn infant showing the typical features of Omenn syndrome, including generalised scaly erythroderma, alopecia, lymphadenopathy and hepatosplenomegaly.

(Courtesy of the Audiovisual Department, University of Newcastle.)

DiGeorge anomaly

This condition results from abnormal cephalic neural crest cell migration into the third and fourth pharyngeal arches in early embryological development. A microdeletion at chromosome 22q11.2 is present in 90% of cases, while the remainder are associated with other chromosomal anomalies, particularly 10p–. Whilst classically recognised by the triad of congenital heart defects, immunodeficiency secondary to thymic hypoplasia and hypocalcaemia secondary to parathyroid gland hypoplasia, an expanded phenotype is increasingly recognised. Conotruncal heart defects are most often associated with the syndrome, but other defects include tetralogy of Fallot, septal defects, pulmonary atresia and aberrant subclavian vessels.

Severe T-lymphocyte immunodeficiency presenting with a SCID phenotype of profound lymphopenia and poor lymphocyte proliferation is rare and accounts for <1.5% of cases. A similar clinical picture has been described in some patients with CHARGE syndrome (coloboma of the eye, heart defects, atresia of the choanae, retardation of growth and/or development, genital and/or urinary abnormalities and ear abnormalities and deafness) ( ).

All children suspected of having DiGeorge syndrome (with or without 22q11.2 deletion) should have an immunological evaluation including lymphocyte subset analysis, T-lymphocyte proliferative responses, immunoglobulin levels and specific antibody responses to vaccination antigens.

Blood products should be irradiated if T-lymphocyte mitogen responses are poor. Patients with severe immune deficiency have been successfully treated with HSCT: thymic transplantation may be a preferred option for some patients, although it is available in only a few centres worldwide ( ). In the partial phenotype with normal T-lymphocyte function, the usual infant vaccination schedule can be followed. It is safe to give live vaccines such as measles, mumps, rubella vaccine or varicella-zoster vaccine as long as the CD4 count exceeds 400 cells/ml, and there are normal T-lymphocyte mitogen responses. Demonstration of good antibody responses to tetanus and Haemophilus influenzae type B vaccination give further reassurance that live vaccines can be safely administered ( ). Prophylactic antibiotics and, occasionally, intravenous immunoglobulin (IVIG) can be helpful, particularly for young children with recurrent infection due to humoral deficiency.

Wiskott–Aldrich syndrome

Immunodeficiency, thrombocytopenia, eczema and an increased risk of autoimmune disorders and malignancy characterise this X-linked recessive condition. Mutations in the same gene are found in patients with X-linked thrombocytopenia and X-linked neutropenia.

Clinical features can be surprisingly mild, especially in the newborn. Thrombocytopenia with a low mean platelet volume (<5 fl) is always present and should raise suspicion of the diagnosis even if bruising and bleeding are minimal.

Acute bleeding episodes may require platelet transfusions (irradiated to prevent GvHD). IVIG, with or without prophylactic antibiotics, reduces infections, and splenectomy may suppress autoimmune phenomena. Without HSCT, the prognosis is variable, but median survival is between 3 and 15 years. The presence or absence of WAS protein correlates with outcome, with patients who have no expressed protein having a worse outcome than those with expressed protein ( ). The efficacy of HSCT for patients with WAS is long established, with good outcomes following related and unrelated donor HSCT ( ).

X-linked hyper-IgM syndrome (CD40 ligand deficiency)

X-linked hyper-IgM syndrome is a combined immunodeficiency caused by a T-lymphocyte defect and not an isolated antibody deficiency, as was previously thought. The defect is in the gene encoding for the CD40 ligand (CD154) expressed on T lymphocytes which activate B lymphocytes and monocyte/macrophage-derived cells. Without CD40L binding, B lymphocytes cannot switch from producing IgM to producing IgA, IgG and IgE. Pulmonary macrophages are not activated, resulting in Pneumocytis jiroveci pneumonia, and ineffective Kupffer cell function allows repeated infection with Cryptosporidium parvum and similar organisms, leading to sclerosing cholangitis, pancreatitis and malignancy. Absent IgA and IgG results in sinopulmonary and invasive bacterial infection. Neutropenia with oral ulceration is common and this, together with low or absent IgA and IgG, and normal or raised IgM, should suggest the diagnosis ( ).

Patients should receive co-trimoxazole, as Pneumocystis carinii pneumonia (PCP) prophylaxis, and immunoglobulin replacement therapy. With adequate replacement, the rising IgM levels may normalise. The neutropenia sometimes responds to granulocyte colony-stimulating factor (G-CSF) and IVIG. All drinking water should be boiled. Azithromycin prophylaxis may lessen the risks of Cryptosporidium parvum . HSCT should be considered for this condition ( ). A much rarer, but similar, AR disease, CD40 deficiency, presents with similar features; treatment is the same as for CD40 ligand deficiency ( ).

Immunodeficiency and short-limbed dwarfism

Cartilage hair hypoplasia is the best described form, inherited in an AR manner and associated with mutations in the RMRP gene, which encodes endoribonuclease Rnase mitochondrial RNA processing (MRP). Severe short-limbed short stature (211.8–22.1 sd ) with X-ray appearances of metaphyseal and spondyloepiphyseal dysplasia are always present, accompanied by sparse light hair in most patients. Severe anaemia and Hirschsprung disease are uncommon but well-recognised associations, as are malignancies, notably lymphoma and skin carcinoma. The immunodeficiency is surprisingly variable; most have T lymphopenia and impaired in vitro mitogen proliferative responses, but only half suffer recurrent infection. However, some have IgA and/or IgG subclass deficiencies, with frequent ear infections. Patients are excessively vulnerable to viral infections, particularly varicella-zoster virus, Epstein–Barr virus and other human herpesvirus infections, and the risk of infective death is 300 times greater than normal. Rare patients can present with a SCID immunophenotype, but no limb dysplasia ( ). Severely affected patients should be assessed for bone marrow transplantation (BMT), which has been successful in correcting the immunodeficiency ( ).

Abnormalities of T- and B-lymphocyte function are seen in a number of other osteochondrodysplasias, including Shwachman syndrome, in which there are neutropenia and pancreatic insufficiency and Schimke immuno-osseous dysplasia, which features radiographic changes of spondyloepiphyseal dysplasia, nephrotic syndrome and cellular immunodeficiency.

Autoimmune lymphoproliferative syndrome

Apoptosis (programmed cell death) regulates immune responses once an infection has been countered, to delete many antigen-specific lymphocytes that clonally expanded to deal with the infection. Apoptosis defects lead to marked autoimmune and lymphoproliferative features which characterise autoimmune lymphoproliferative syndrome (ALPS) ( ).

Chronic mucocutaneous candidiasis

Chronic mucocutaneous candidiasis (CMC) describes a heterogeneous group of primary or secondary disorders characterised by chronic infection of skin, nails and mucous membranes by organisms of the genus Candida , most commonly Candida albicans. The precise molecular defect is not known for most forms of primary CMC, although defects in cytokine signalling are apparent in some patients ( ; ). Recurrent and persistent Candida of the mouth, napkin area, skins and nails is the hallmark of this condition, but the severity varies considerably and invasive disease almost never occurs. Failure of usually effective antifungal drugs to clear Candida distinguishes primary CMC from other conditions that predispose to Candida, such as secondary immunodeficiency, steroid treatment or systemic antibiotics. Candidiasis is usually first noticed early in infancy and in severe cases gross oesophageal involvement causes dysphagia, gastro-oesophageal reflux and failure to thrive, whilst skin lesions may be extremely disfiguring and distressing. Cases may be familial or sporadic with recessive or dominant patterns of inheritance.

Treatment with azole antifungals, such as fluconazole, can be very effective, even in severe cases, but may not completely eradicate infection, and infection often recurs on stopping treatment.

IPEX syndrome

Immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome is characterised by early-onset type 1 insulin-dependent diabetes mellitus, infantile ichthyosiform dermatitis, protracted diarrhoea and severe total parenteral nutrition-dependent enteropathy and thyroiditis. Mutations in the FOXP3 gene have been described in affected patients ( ).

Hoyeraal–Hreidarsson syndrome

This X-linked disorder is characterised by microcephaly, cerebellar hypoplasia, aplastic anaemia and growth retardation. A progressive combined immunodeficiency, with hypogammaglobulinaemia and lymphopenia, is a well-recognised association.

ICF syndrome

Immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome is an AR disorder characterised by structural chromosomal abnormalities in chromosomes 1, 9 and 16 in lymphocytes, easily seen on chromosome analysis. Other cells do not show these changes. Affected children develop severe recurrent infections and have immunoglobulin deficiency, often with agammaglobulinaemia but with normal numbers of T and B lymphocytes ( ). T-lymphocyte immunity is not normal and affected children can present with Pneumocystis carinii infection in infancy; severe viral warts and cutaneous fungal infection also occur. HSCT may be curative for selected patients.

Neutropenia

Neutropenia may result from reduced production in the bone marrow or from increased peripheral destruction, distinguished by the findings on bone marrow examination. It most frequently follows decreased production induced by disease processes or drug treatments, as well as sepsis in preterm or stressed neonates. Increased consumption may occur in autoimmune states, including neonatal alloimmune neutropenia and those associated with immune deficiency. The degree of neutropenia will influence the clinical picture: neutrophil counts of less than 0.5 × 10 ⁹ /l carry a major risk of infection, whilst counts below 0.2 × 10 ⁹ /l are associated with a significant risk of life-threatening sepsis.

Severe congenital neutropenia (Kostmann’s syndrome)

This AR disease was originally described by . Onset is within the first year of life with recurrent and life-threatening infections. Symptoms include cellulitis, perirectal abscesses, peritonitis, stomatitis and meningitis. Bone marrow examination shows an arrest at the promyelocyte to myelocyte maturation stage. Kostmann’s syndrome is due to mutations in Hax1, but defects in other genes can lead to a similar clinical picture ( ).

Shwachman–Diamond syndrome

This rare AR disorder is characterised by exocrine pancreatic insufficiency, skeletal abnormalities, bone marrow dysfunction and recurrent infections. Neutropenia occurs in all patients, whilst 10–25% of patients also have pancytopenia. There is an increased incidence of malignancy.

Treatment of neutropenia with G-CSF results in increased counts and fewer infections in almost all patients with neutropenia not secondary to peripheral destruction. Concerns about the induction of leukaemias with prolonged use have not been realised, although pretreatment and annual bone marrow aspirates are recommended. BMT may be indicated in selected cases.

Haemophagocytic lymphohistiocytosis

Congenital neutropenia or neutrophil dysfunction, with or without associated hypopigmentation, is a feature of diseases with defects in proteins that control lysosomal secretion in melanocytes and immune cells. An important presentation of these diseases is haemophagocytic lymphohistiocytosis (HLH), a fatal inflammatory disease characterised by high swinging fevers, hepatosplenomegaly, jaundice and erythematous rash, respiratory distress and pancytopenia, consequent to hypercytokinaemia and organ infiltration by CD8+ lymphocytes and macrophages. Patients appear septic but blood cultures are usually sterile. Laboratory findings include an acute-phase response, elevated ferritin and elevated fasting triglycerides. Examination of bone marrow, cerebrospinal fluid, pleural effusions or ascitic fluid may demonstrate haemophagocytosis. Haemophagocytosis may occur secondary to a number of infections, in particular viral infections, and careful exclusion of infections by serology/polymerase chain reaction should be undertaken. Haemophagocytosis is seen in a number of immunodeficient states, including Chédiak–Higashi syndrome, Griscelli syndrome and X-linked lymphoproliferative disease (XLP) ( Table 39.2 ). Chédiak–Higashi syndrome is a rare AR disease with partial oculocutaneous albinism, recurrent bacterial infections and a high risk of developing HLH, which is usually fatal. Variable neurological manifestations are also recognised. Characteristic giant lysosomal granules are seen in the cytoplasm of all cells containing these organelles, and are easily detected on a peripheral blood film. Individuals with Griscelli syndrome resemble those with Chédiak–Higashi syndrome in that they have variable hypopigmentation of the skin and hair and recurrent pyogenic infections associated with absent delayed-type cutaneous hypersensitivity and impaired NK cell function. Hypogammaglobulinaemia can be seen as a secondary phenomenon. In contrast to Chédiak–Higashi syndrome, large lysosomal granules are not seen, and examination of the hair by electron microscopy shows large clumps of pigment, with the accumulation of mature melanosomes in melanocytes. Neurological abnormalities are absent in these patients. They are also at high risk of developing macrophage activation syndrome unless treated by BMT.

Leukocyte adhesion deficiency

Inherited defects in leukocyte cell surface molecules can prevent their egress into infected tissue, resulting in a characteristic clinical presentation. In leukocyte adhesion deficiency type I, deficiency of the 95-kD β chain (CD18), common to the β2 integrin family of cell surface adhesive molecules, leads to a profound immunodeficiency affecting the function of neutrophils, monocytes and certain lymphocytes, including T and NK cytotoxic cells ( ). Inheritance is AR. Chemotaxis, adherence and phagocytosis are markedly depressed. The phenotype varies according to the severity of the mutation. Leukocytes are attracted to areas of infection and become fixed to the vessel walls at sites of inflammation in the usual way but cannot pass out into the tissues, causing infarction of small vessels and rapidly expanding necrotic lesions without pus.

Individuals with the most severe phenotype (<1% expression) present in the first weeks of life with delayed umbilical cord separation (the cord fails to shrink down and may not separate until 3–4 weeks of age) and rapidly progressive erosive perianal ulcers. Delayed umbilical cord separation does not occur in patients with some expression of the molecule (usually in the range 2–10% of normal expression). In all forms there is excessive susceptibility to bacterial and fungal infections. Gingivitis and periodontitis are common, and more deep-seated infections of bone, respiratory tract and gastrointestinal tract are often seen. Non-infective inflammatory lesions, particularly affecting the skin and resembling pyoderma gangrenosum, can occur in the partial forms of the deficiency and are often responsive to steroids.

There is almost always a neutrophilia (because of failure of the cells to migrate out of the circulation) and a profound neutrophil chemotactic defect. Diagnosis is confirmed by demonstrating the absence of the cell surface markers recognised by anti-CD11/CD18 monoclonal antibodies.

In the severe form, early death from infection occurs unless a successful BMT can be performed. In the partial forms, supportive and expectant management is pursued in the first instance, but BMT may become necessary.

Hyper-IgE syndromes

These complex disorders are characterised by extreme elevation of the serum IgE level (usually in the range 2000–40 000 U/l), chronic dermatitis and repeated lung and skin infections ( ). Patients present in infancy with eczema, but in the neonatal period a vesicular rash may be present in the first few days of life. Staphylococcus aureus , the predominant pathogen, can cause pneumatoceles and chronic lung damage. Recurrent candidiasis may be a feature. Treatment is long-term antistaphylococcal antibiotic prophylaxis, usually with flucloxacillin. Three genes have been identified, leading to the clinical picture. Defects in STAT3 give rise to the classical autosomal-dominant clinical picture, whereas AR defects in TYK2 and DOCK8 are associated with severe molluscum contagiosum and herpesvirus infection.

Anhydrotic ectodermal dysplasia, incontinentia pigmenti and defects in the NEMO gene

Until recently, incontinentia pigmenti and X-linked anhydrotic ectodermal dysplasia were thought to be unrelated disorders. Incontinentia pigmenti is a rare X-linked dominant condition characterised by developmental abnormalities of the skin, hair, teeth and central nervous system. Affected female infants present with a characteristic rash, occurring in four phases of erythema and vesicles, hyperkeratosis, hyperpigmentation and finally pallor and atrophy. Previously thought lethal in male fetuses carrying the affected X chromosome, rare male infants with a progressive combined immunodeficiency have been described. Patients with X-linked anhydrotic ectodermal dysplasia present with sparse scalp hair, conical teeth and absent sweat glands. Some suffer from recurrent sinopulmonary infection, often with encapsulated organisms, and have poor antibody responses to polysaccharide antigens, or frank hypogammaglobulinaemia. Hypofunctional mutations in the NEMO gene encoding a protein required to activate the transcription factor NF-κB have been described in male patients with both incontinentia pigmenti and X-linked anhydrotic ectodermal dysplasia, suggesting that these conditions represent variants of the same disorder ( ). Other proteins in the same signalling pathway can cause a similar clinical picture, although inheritance is AR.

Late-onset sepsis

Late-onset infection in neonates is defined as infection becoming clinically evident more than 48–72 hours after birth, and is usually the result of nosocomially acquired organisms, hence the term ‘healthcare-acquired infection’.

Group B streptococcus

GBS is an important cause of serious infection in neonates, infants and immunocompromised or pregnant adults. The overall incidence of GBS infection in the UK is 0.72/1000 live births, though there are regional variations, with the highest incidence being in Northern Ireland and the lowest in Scotland ( ). GBS is a commensal of the gastrointestinal tract and vagina, and colonises some 10–30% of pregnant women in the UK. There are numerous identifiable serotypes of GBS, based on capsular polysaccharide antigens: Ia, Ib, II, III, IV, V, VI, VII and VIII. All are implicated in early-onset disease, with national variations in prevalence. In the UK, enhanced surveillance has demonstrated that GBS serotypes III, Ia and V are the most commonly identified isolates causing early-onset infection ( ). Simultaneous infection with two distinct serotypes has been described ( ). In addition to the polysaccharide antigens there are surface-exposed protein antigens which may also contribute to the pathogenicity of particular strains ( ).

It is well established that GBS can cross intact fetal membranes. While prolonged rupture of fetal membranes (>18 hours) is a feature in 44% of newborns with invasive GBS infection, for 56% of affected babies, membrane rupture occurred less than 18 hours before birth ( ). Although GBS infection is uncommon, mortality and morbidity are high, especially for preterm babies ( ). This may partly be a consequence of the acute inflammatory response to GBS. GBS is able to invade pulmonary endothelial cells, leading to release of eicosanoids such as prostacyclin and prostaglandin E2 ( ). In animal models, GBS causes a dose-dependent increase in pulmonary arterial pressure and pulmonary and systemic vascular resistance, and decreases cardiac output and heart rate. The quantitative response of the pulmonary vascular resistance is GBS strain-dependent in animal models, with serotype Ib having a greater effect than serotype III ( ). Postmortem examination demonstrates that many infants develop alveolar hyaline membranes, as well as pneumonia ( ). In the preterm infant, this is likely to represent coexistent surfactant-deficient hyaline membrane disease of prematurity ( ), but in term babies the hyaline membranes are a non-specific change associated with GBS infection ( ), and may reflect a secondary surfactant insufficiency.

Most incidence figures are based on ‘culture-proven’ GBS whereby GBS is grown in blood or cerebrospinal fluid (CSF). Various factors may decrease the yield of blood and CSF cultures in the presence of true infection. Blood cultures are often negative in pneumonia ( ). Other possible explanations include prior use of antibiotics (e.g. when given to the mother as IAP) ( ), inadequate blood volumes obtained ( ) or low-level bacteraemia. In the case of GBS, ‘probable’ infection has been defined as an episode of clinical sepsis likely to have been caused by GBS, and indicated by GBS surface colonisation together with clinical features of sepsis but with negative blood cultures. Prospectively collected data in a UK tertiary neonatal unit from all neonates requiring a sepsis screen in the first 72 hours of life demonstrated a combined rate of definite and probable GBS infection of 3.6/1000 live births ( ). This suggests a much greater disease burden in the UK than the incidence based on culture-proven sepsis.

Prior to the introduction of a national GBS-screening programme and IAP, the reported prevalence of early-onset GBS sepsis in the USA was 1.8 per 1000 live births ( ); in Canada, 1.75 per 1000 ( ); in Australia, 1.3 per 1000 ( ); and in Spain, 1.2 per 1000 ( ). The prevalence in Finland is around 0.76 per 1000 ( ).

Risk factors for early-onset GBS infection can be identified in up to 60% of cases and include preterm delivery (<37 weeks), prolonged rupture of membranes (PROM ≥ 18 hours) or known genital carriage of GBS during pregnancy ( ), although GBS can cross intact membranes. Infection is the most common identifiable reason for spontaneous vaginal preterm delivery, so preterm delivery alone represents a risk factor for infection. The overall mortality is approximately 10% but is significantly higher among infants born prematurely ( ). Up to 7% of survivors of GBS infection may suffer some disability; the risk is obviously higher if there is meningitis. Opportunities to prevent GBS infection using a risk factor based approach to GBS-prevention are often missed ( ).

The mode of presentation and frequency of infection differ according to age at onset: GBS most commonly causes early-onset disease, defined as being <7 days (0.47/1000), manifesting as sepsis or pneumonia. Late-onset GBS disease can occur from 7 days to 3 months of life and is less common (0.25/1000); the more frequent presentation is with meningitis and sepsis. Late-onset GBS infection may result from invasion of colonising organisms acquired at birth or nosocomially or possibly from breast milk ( ; ).

Presentation of GBS infection

Nearly 90% of neonates with early-onset GBS have signs of infection within 12 hours of delivery ( ; ). Early stages of infection may be very subtle. A baby may simply not be feeding well or be excessively sleepy. Tachypnoea, apnoea or other respiratory distress are the most common presenting signs ( ; ). GBS sepsis may alternatively present with respiratory failure, cyanosis and shock, and the baby’s condition at delivery may be clinically indistinguishable from perinatal hypoxic ischaemia. GBS infection should always be suspected in a baby who has more severe respiratory distress syndrome (RDS) than would have been anticipated, as RDS and GBS pneumonia are radiologically indistinct. In severe cases, persistent pulmonary hypertension of the newborn, hypotension, metabolic acidaemia, tachycardia and poor peripheral perfusion may develop and are poor prognostic features.

If infection is suspected in a baby, the baby should be promptly investigated (including a chest radiograph (CXR)), and treated with antibiotics. C-reactive protein (CRP) measurements may be normal in the early stages of infection ( ) as it takes at least 12 hours for a CRP level to become raised ( ). A normal CRP does not exclude GBS infection, but may rise after 12–24 hours in infection. A raised CRP may be due to infection or other causes of inflammation. Some use the CRP to monitor the response to therapy, but there is no evidence that this is superior to clinical response.

Benzylpenicillin is the antibiotic of choice for GBS. Any organisms present in blood usually require a minimum of 48 hours to culture. During this time babies are treated with antibiotics until the results of the cultures are known. It is not uncommon for a baby to have signs of infection but for the tests on blood, swabs or CSF to be negative. Pneumonia is the most common focal infection caused by GBS, and, as already stated, can mimic RDS radiologically, with bilateral ground-glass shadowing and air bronchograms ( Fig. 39.7 ). Signs of pneumonia may be minimal and only manifest on a radiograph and with clinical signs and raised inflammatory markers (raised CRP, alterations in absolute neutrophil count (ANC) and low platelet count). It is common practice to treat such babies with antibiotics for many days because of the difficulty in being certain about whether or not bacteria are causing their illness ( ). As a result not only do many infants receive antibiotics that are ultimately not required, but they may also receive them for a long time. In the prospective study referred to above ( ), <5% of infants treated for infection had either culture-proven or culture-negative clinical sepsis; thus >95% of babies receiving antibiotics did not need them. Excessive antibiotic use may alter antibiotic susceptibilities as well as the types of colonising microbial flora, especially in the gut. Possible consequences include the emergence of antibiotic resistance in common bacterial pathogens, such as E. coli ( ; ; ), the emergence of new pathogens, such as Enterobacter sp. ( ), and an increased risk of atopy and asthma in childhood ( ; ; ).

Chest radiograph of baby with culture-proven group B streptococcus and more severe respiratory distress syndrome then was anticipated.

Listeria

Listeria monocytogenes is a short Gram-positive intra- and extracellular rod. The most important reservoir for transmission to humans is probably food, especially dairy products, contaminated by infected farm animals ( , ). Neonatal infection in the UK is now uncommon ( Table 39.4 ). Women infected with HIV are considerably more susceptible to L. monocytogenes than are the general population ( ). Three main types of fetal and neonatal infection are found: (1) transplacental infection; (2) early-onset infection acquired intrapartum; and (3) late-onset infection, usually meningitis, probably due to nosocomial infection.

Enterobacteriaceae

The family Enterobacteriaceae includes many genera and species ( Table 39.6 ). Members are Gram-negative, facultatively anaerobic bacilli, which may appear as coccobacilli on Gram stain. The virulence of these species is controlled by a number of factors, including the ability to adhere, colonise, invade tissues and produce various toxins. Different antigens can be used to differentiate different serologic groups. The capsular antigen K1, present in certain strains of E. coli , has been the most documented virulence factor associated with E. coli meningitis and sepsis. These strains are distinct from those associated with diarrhoeal disease. Some Enterobacteriaceae harbour plasmids providing antimicrobial resistance genes. An increasing number of E. coli and Klebsiella strains produce plasmid-mediated extended-spectrum β-lactamases, which can inactivate cephalosporins, penicillins and aztreonam. Family members are ubiquitous in nature but almost all share the gastrointestinal tract as a common niche, and, apart from Yersinia , Shigella and Salmonella , can be normal resident flora if confined to their natural environment. E. coli is so commonly isolated from colonic flora that it is used as a primary marker of faecal contamination in water purification. Although Enterobacteriaceae can cause opportunistic infection, they are often harmless commensals. Members are broadly divided into opportunistic and primary pathogens. Opportunistic pathogens are often part of the normal faecal flora. They may produce serious extraintestinal opportunistic infections, however; for example E. coli is a member of the normal bowel flora but can cause urinary tract infections (UTIs), septicaemia and meningitis. Primary pathogens include Salmonella , Shigella and Yersinia , and are not part of normal gut flora. They are true pathogens and will cause infections if ingested.

Gram-negative bacteraemia and antimicrobial non-susceptible Enterobacteriaceae

The Centers for Disease Control National Nosocomial Infection Surveillance System has reported a substantial increase in third-generation cephalosporin-resistant enteric Gram-negative bacilli causing nosocomial infections in intensive care units ( ; ). Gram-negative bacteraemia in children has also been positively associated with the use of antibiotics in addition to cephalosporins, including aminoglycosides and vancomycin, but also correlates with surgical interventions, central venous catheters, parenteral nutrition, antacids and dexamethasone. The strongest association in one study was with the use of vancomycin ( ), hence the recommendation that vancomycin should not be used for empirical treatment on the basis of suspicion of bacteraemia.

ELBW babies, <1000 g, who are colonised with antimicrobial non-susceptible Enterobacteriaceae (ANE) and those who receive prolonged exposure to antimicrobial agents are at increased risk of ANE infections ( ). Such ANE include E. coli, Enterobacter spp., Citrobacter spp., Klebsiella spp. and Serratia spp. They are defined as being ‘members of the Enterobacteriaceae family exhibiting non-susceptibility to ceftazidime or with evidence of extended spectrum beta-lactamase production’. Multiply resistant Enterobacteriacaeae strains (MRE, defined as ‘strains resistant to three or more classes of antibiotic’), have been found at high frequency in NICU infants, and persist in a significant proportion of infants for at least 6 months, even after discharge from hospital ( ). Such high prevalence of colonisation increases the probability of MRE infection in vulnerable infants and outbreaks are increasingly described within NICUs ( ; ; ; ; ; ).

Colonisation with MRE strains has been detected as early as on the first day of hospitalisation ( ), and may follow transmission from other NICU patients, or may evolve endogenously from selective pressures exerted by antimicrobial therapy.

In the UK, the majority of pathogens, other than CoNS, causing EOS (>94%) are susceptible to commonly used empiric first-line antibiotic regimens of penicillin/gentamicin or amoxicillin, amoxicillin combined with cefotaxime or cefotaxime monotherapy ( ; ). More than 95% of organisms causing LOS are susceptible to gentamicin with either flucloxacillin ( ; NeonIN), or amoxicillin and amoxicillin with cefotaxime, but only 79% are susceptible to cefotaxime monotherapy ( ). This supports the use of narrow-spectrum antimicrobials in the form of penicillin/gentamicin as ‘first-line’ therapy for EOS and flucloxacillin/gentamicin for LOS, with use of vancomycin restricted to situations where there is a serious risk of CoNS infection.

Supoptimal empiric antibiotic therapy potentially leads to worse outcomes ( ; ), but must be balanced against the hazards of routine use of broad-spectrum antibiotics for empiric therapy, since administration of these correlates with the development of resistant organisms ( ; ).

Enterococci

Previously classified as group D streptococci, enterococci are Gram-positive cocci that are natural inhabitants of the human intestinal tract. They have a survival advantage over other organisms in that they can grow in extreme conditions and are resistant to multiple antimicrobial agents. Enterococci are associated with nosocomial infection, most commonly bacteraemia and UTI. Prolonged hospitalisation, immunocompromise and prior surgical procedures are risk factors. Because of intrinsic or acquired resistance to several antimicrobial agents, including aminoglycosides, beta-lactams and glycopeptides, e.g. vancomycin and teicoplanin, differentiation of enterococci from other streptococci and susceptibility testing are important.

History

It is important to ask specific questions about maternal, perinatal and neonatal events. Apparently trivial episodes of pyrexia or skin rash may be significant, as well as contact with known cases of infectious disease; whether a mother was unwell or febrile; had a tender uterus or a vaginal discharge; the exact time of membrane rupture; potentially infectious lesions on her cervix or vulva; presence of a cervical suture; fetal tachycardia; raised maternal CRP or white cell count. The smaller and more preterm a baby, the higher the risk of infection and mortality ( ).

If the mother has overt intrapartum infection, 15.2% of preterm babies and 4.1% of term babies will develop infection. Maternal UTI is an important risk factor for neonatal sepsis.

Factors predisposing to nosocomial infection include a history of possible contact with an infected person or environment, not receiving enteral feeds ( ; ), indwelling catheters or receiving TPN ( ; Shinwell et al. 2004). Babies who have had gut surgery or gut-related problems also have a significant risk of nosocomial sepsis. Infection is more likely to occur in a baby in intensive care, and infection will prolong hospital stay and morbidity ( ; ; ; ).

Signs of neonatal sepsis

In the early stages, signs may be subtle and, although difficult to define, a mother or nurse may report that a baby is simply ‘ not right’. Isolated tachypnoea (respiratory rate sustained above 60 breaths/min and slight recession) and feeding difficulties (not feeding or poor suck) are the most frequent early signs of infection.

Microbiological investigation

Blood culture remains the ‘gold standard’ for diagnosing infection, though it has limitations ( ). The majority of significant blood cultures are positive by 48 hours ( ).

Collecting blood for culture

Blood for culture should be drawn from a freshly punctured blood vessel using strict aseptic technique and a closed system. The greater the volume of blood, the higher the yield of organisms, but 0.5 ml of blood is usually sufficient for a successful culture ( ). Taking two cultures from separate sites reduces false-positive diagnoses of infection.

Antibiotic therapy should be stopped after 36–48 hours if cultures are negative and there are no further signs of infection. Alternatively, when there is a suspicion that clinical progress is suboptimal, consideration should always be given to an empiric change of antibiotic therapy to include a broader spectrum of pathogens. Such analyses should be validated in other data sets but have the potential to improve neonatal outcome by ensuring appropriate antibiotics are used as early as possible. Once a bacterium is identified, the antibiotic regimen should be targeted appropriately.

Surface swabs, tracheal secretions, endotracheal tube tip culture and gastric aspirates

Routine swabbing of sites such as the umbilicus, groin, ear, nose, throat, pharynx and rectum are informative about colonisation, but numerous studies have shown that the results of surface cultures are of limited value in diagnosis ( ; ). Colonisation of babies, for example of endotracheal secretions or of skin and mucosal surfaces, without clinical signs of infection does not warrant antibiotic treatment ( ). The same applies to gastric aspirates and maternal vaginal swabs which are indicative of colonisation but not invasive infection. The latter may be helpful in a baby who is symptomatic of infection if positive for GBS.

Urine

A UTI is defined by there being a pure growth of at least 10 ⁸ organisms per litre of urine (10 ⁵ /ml) in a specimen of urine. In order to avoid the problems of contamination of urine specimens, suprapubic aspiration of urine or fresh catheter urine specimens should be obtained in the initial assessment of the sick baby who is thought to be septicaemic, though this may be impracticable. A clean-catch sample may be helpful if the genitalia have been adequately cleaned. The only value of ‘bag urines’ is that if they are clear they exclude UTI. Positive bag urines, even when the genitalia have been adequately cleaned, should always be viewed with suspicion, even in the presence of white cells, as babies may have sterile pyuria.

Vascular lines and thoracocentesis tubes

The tips of umbilical cannulae, central lines and thoracocentesis tubes are usually sent for culture, though they have a limited role for diagnosing infection as they are indicative of colonisation, including of the skin through which they are withdrawn.

Lumbar puncture

A lumbar puncture should be performed as part of the sepsis screen in most ill babies before antibiotics are started. Exemptions include babies with acute RDS ( ), pulmonary infection complicating long-term intermittent positive-pressure ventilation (IPPV), babies with overt localised infection, such as NEC or pneumonia, and babies who would not be able to tolerate the procedure ( ). A low platelet count is a contraindication to lumbar puncture; if the investigation is essential a platelet transfusion should be given first. Asymptomatic term babies undergoing investigation because of risk factors for sepsis do not require a lumbar puncture, nor is jaundice requiring treatment in a well baby an indication for lumbar puncture ( ; ). Meningitis is more frequently a feature of LOS than EOS ( ; ).

Lumbar puncture must be performed with strict sterile precautions, avoiding excessive flexion of the trunk, which may otherwise cause respiratory embarrassment or apnoea. It is safer to intubate and ventilate a very unstable baby before proceeding.

CSF analysis

High white blood cell counts in CSF have sometimes been reported in babies without meningitis, but a polymorphonuclear count higher than 20/mm ³ should be regarded with suspicion ( ), and counts above 30/mm ³ are strongly indicative of meningitis ( Appendix 8 ). For blood-stained CSF the ratio of red cells to white cells should be 500 : 1 in uninfected CSF.

The upper normal limit of CSF protein is 1.5–2.0 g/l in the term baby ( Appendix 8 ). Preterm babies may have higher CSF protein concentrations but rarely greater than 3.0 g/l. Protein levels are usually raised in meningitis. CSF glucose levels should be compared to a simultaneous measurement of blood glucose, and should be at least 50% of the blood glucose level. A low level, less than 30% of the blood glucose, suggests bacterial meningitis.

The CSF should be Gram-stained, examined under a microscope, and cultured for bacteria. Other tests which may be indicated include PCR tests for GBS, herpesvirus and enteroviruses.

Radiology

All babies with suspected sepsis should have a CXR. An abdominal radiograph may also be helpful in the differentiation between septic ileus and NEC. Radiographs may not detect bone or joint infection in the early stages.

Haematological investigation

Acute-phase proteins

Cytokines

Immunological tests

Genetic techniques

It is now possible to amplify highly conserved DNA sequences from a variety of Gram-positive and Gram-negative organisms, as well as many viruses, using PCR, while avoiding the simultaneous amplification of associated human DNA. This method has the potential to be automated and to provide rapid diagnosis of bacteraemia.

In a meta-analysis of various intrapartum group B streptococcus colonisation tests, the most accurate was the real-time PCR test, although it took 10 minutes longer to perform PCR as compared to the Optical immunoassay (OIA) test (40 versus 30 minutes) ( ) ( Box 39.2 ).

Box 39.2

Additional investigations if fungal infection is suspected or diagnosed

• •
  

  End-organ evaluation to include ( ):

  
  
  
  
  • •
    

    Abdominal ultrasound (including renal ultrasound)

    
    
  • •
    

    Cerebral ultrasound

    
    
  • •
    

    Lumbar puncture

    
    
  • •
    

    Fundoscopy

    
    
  • •
    

    Echocardiogram

  
  
  
  
• •
  

  Blood cultures at 24–48-hourly intervals to ensure clearance

  
  
• •
  

  Suprapubic or catheter specimen of urine

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