Assessment of infant position and timing of stylet removal to improve lumbar puncture success in neonates (NeoCLEAR): an open-label, 2 × 2 factorial, randomised, controlled trial.

BACKGROUND: Newborn infants are the highest-risk age group for bacterial meningitis. Lumbar punctures are therefore frequently performed in neonates, but success rates are low (50-60%). In Neonatal Champagne Lumbar punctures Every time-A Randomised Controlled Trial (NeoCLEAR), we sought to optimise infant lumbar puncture by evaluating two modifications to traditional technique: sitting position versus lying down and early stylet removal (stylet removal after transecting the subcutaneous tissue) versus late stylet removal. METHODS: NeoCLEAR was an open-label, 2 × 2 factorial, randomised, controlled trial, conducted in 21 UK neonatal and maternity units. Infants requiring lumbar puncture at 27+0 to 44+0 weeks corrected gestational age and weighing 1000 g or more were randomly assigned (1:1:1:1) to sitting position and early stylet removal, sitting position and late stylet removal, lying position and early stylet removal, or lying position and late stylet removal using a 24/7, web-based, secure, central randomisation system. Block randomisation was stratified within site by corrected gestational age (27+0 to 31+6 weeks, 32+0 to 36+6 weeks, 37+0 to 40+6 weeks, or 41+0 to 44+0 weeks), using variable block sizes of four and eight with equal frequency. Laboratory staff were masked to allocation. The primary outcome was successful first lumbar puncture, defined as obtaining a cerebrospinal fluid sample with a red blood cell count of less than 10 000 cells per µL. The primary and secondary (including safety) outcomes were analysed by the groups to which infants were assigned regardless of deviation from the protocol or allocation received, but with exclusion of infants who were withdrawn before data collection or who did not undergo lumbar puncture (modified intention-to-treat analysis). This study is registered with ISRCTN, ISRCTN14040914. FINDINGS: Between Aug 3, 2018, and Aug 31, 2020, 1082 infants were randomly assigned to sitting (n=546) or lying (n=536), and early (n=549) or late (n=533) stylet removal. 1076 infants were followed-up until discharge and included in the modified intention-to-treat analysis. 961 (89%) infants were term, and 936 (87%) were younger than 3 days. Successful first lumbar puncture was more frequently observed in sitting than in lying position (346 [63·7%] of 543 vs 307 [57·6%] of 533; adjusted risk ratio 1·10 [95% CI 1·01 to 1·21], p=0·029; number needed to treat=16). Timing of stylet removal had no discernible effect on the primary outcome (338 [62·0%] of 545 infants in the early stylet removal group and 315 [59·3%] of 531 in the late stylet removal group had a successful first lumbar puncture; adjusted risk ratio 1·04 [95% CI 0·94-1·15], p=0·45). Sitting was associated with fewer desaturations than was lying (median lowest oxygen saturations during first lumbar puncture 93% [IQR 89-96] vs 90% [85-94]; median difference 3·0% [2·1-3·9], p<0·0001). One infant from the sitting plus late stylet removal group developed a scrotal haematoma 2 days after lumbar puncture, which was deemed to be possibly related to lumbar puncture. INTERPRETATION: NeoCLEAR is the largest trial investigating paediatric lumbar puncture so far. Success rates were improved when sitting rather than lying. Sitting lumbar puncture is safe, cost neutral, and well tolerated. We predominantly recruited term neonates younger than 3 days; other populations warrant further study. Neonatal lumbar puncture is commonly performed worldwide; these results therefore strongly support the widespread adoption of sitting technique for neonatal lumbar puncture. FUNDING: UK National Institute for Health and Care Research.

Study protocol: NeoCLEAR: Neonatal Champagne Lumbar punctures Every time - An RCT: a multicentre, randomised controlled 2 × 2 factorial trial to investigate techniques to increase lumbar puncture success.

BACKGROUND: The neonatal period carries the highest risk of bacterial meningitis (~ 1 in 5000 births), bearing high mortality (~ 10%) and morbidity (20-50%) rates. Lumbar puncture (LP) remains essential to the diagnosis of meningitis. Though LP is a common procedure in neonates, success rates are lower (50-60%) than in other patient populations. None of the currently-practised neonatal LP techniques are supported by evidence from adequately-powered, randomised controlled trials (RCTs). NeoCLEAR aims to compare two modifications to the traditional technique which are free, accessible, and commonly practised: sitting (as opposed to lying) position, and 'early' (as opposed to 'late') stylet removal. METHODS/DESIGN: Written parental informed consent permitting, infants in neonatal/maternity wards, of 27+ 0 to 44+ 0 weeks corrected gestational age and weighing ≥1000 g, who require an LP, will be randomly allocated to sitting or lying position, and to early or late stylet removal. The co-primary objectives are to compare success rates (the proportion of infants with cerebrospinal fluid red cell count < 10,000/mm3 on first LP procedure) in 1020 infants between the two positions, and between the two methods of stylet removal. Secondary outcomes relate to LP procedures, complications, diagnoses of meningitis, duration of antibiotics and hospital stay. A modified intention-to-treat analysis will be conducted. DISCUSSION: Two modifications to the traditional LP technique (sitting vs lying position; and early vs late stylet removal) will be simultaneously investigated in an efficient and appropriately-powered 2 × 2 factorial RCT design. Analysis will identify the optimal techniques (in terms of obtaining easily-interpretable cerebrospinal fluid), as well as the impact on infants, parents and healthcare systems whilst providing robust safety data. Using a pragmatic RCT design, all practitioners will be trained in all LP techniques, but there will inevitably be variation between unit practice guidelines and other aspects of individual care. An improved LP technique would result in: • Fewer uninterpretable samples, repeated attempts and procedures • Reduced distress for infants and families • Decreased antibiotic use and risk of antibiotic resistance • Reduced healthcare costs due to fewer procedures, reduced length of stay, shorter antibiotic courses, and minimised antibiotic-associated complications TRIAL REGISTRATION: ISRCTN14040914. Date assigned: 26/06/2018.

Immunological effect of administration of sequential doses of Haemophilus influenzae type b and pneumococcal conjugate vaccines in the same versus alternating limbs in the routine infant immunisation schedule: an open-label randomised controlled trial.

BACKGROUND: The use of different limbs for the administration of sequential doses of an intradermal rabies vaccine was shown to result in reduced vaccine immunogenicity. We aimed to assess whether this phenomenon also occurs with routine infant vaccines. METHODS: In this open-label, randomised, controlled study, eligible healthy infants 6-12 weeks of age recruited through five clinical trials units (four in the UK and one in Malta) were randomly assigned in a 1:1 ratio to two vaccination groups: consistent limb or alternating limb. Infants in the consistent limb group received the diphtheria-tetanus-acellular pertussis-inactived polio-Haemophilus influenzae type b combined vaccine (DTaP-IPV-Hib) at 2, 3, and 4 months of age, and the pneumococcal conjugate vaccine (PCV13) at 2, 4, and 12 months, all administered to the right leg. Infants in the alternating limb group received DTaP-IPV-Hib in the left leg at 2 months and in the right leg at 3 and 4 months; and PCV13 in the left leg at 2 months, in the right leg at 4 months, and in the left arm at 12 months. All infants in both groups received the combined H influenzae type b and capsular group C Neisseria meningitidis tetanus toxoid conjugate vaccine (Hib-MenC-TT), administered in the left leg at 12 months. Randomisation was achieved by randomly generated codes, with permuted block size of 30, and was stratified by study site. Group allocation was not masked from study staff and parents of participants after enrolment, but group allocation was masked from laboratory staff assessing blood samples. The current study was a prespecified secondary objective of a parent phase 4 trial that assessed the induction of immunity following varying schedules of vaccination with glyco-conjugate capsular group C Neisseria meningitidis (Men C) vaccines in infancy. The objective of the current study was to compare the immunogenicity and reactogenicity of vaccines delivered in either consistent or alternating limbs. Immunogenicity was assessed by comparing serum IgG geometric mean concentrations at 5, 12, 13, and 24 months, analysed per protocol. This study is registered with ClinicalTrials.gov, number NCT01129518. FINDINGS: Between July 5, 2010, and Aug 1, 2013, we enrolled 509 infants and randomly allocated them to the consistent limb group (n=254) or the alternating limb group (n=255). Anti-H influenzae type b anti-polyribosylribitol phosphate IgG geometric mean concentrations were lower in the consistent limb group than in the alternating limb group at 5 months (consistent limb 0·41 µg/mL [95% CI 0·31-0·54] vs alternating limb 0·61 µg/mL [0·45-0·82]; p=0·0268) and at 12 months (0·35 µg/mL [0·28-0·43] vs 0·50 µg/mL [0·40-0·62]; p=0·0136). Anti-tetanus toxoid antibody IgG geometric mean concentrations were lower in the consistent limb group (1·63 IU/mL [95% CI 1·40-1·90]) than in the alternating limb group (2·30 IU/mL [1·97-2·68]) at 13 months (p=0·0008) and at 24 months (0·44 IU/mL [0·37-0·52] vs 0·61 IU/mL [0·51-0·73]; p=0·0074). Anti-pneumococcal IgG geometric mean concentrations were similar between both groups at all timepoints. The proportions of participants who had adverse events did not differ between the two groups. INTERPRETATION: Use of different (alternating) limbs for sequential doses of routine infant vaccines does not reduce, and might enhance, immunogenicity. The underlying mechanism for this finding warrants further research. FUNDING: NIHR Oxford Biomedical Research Centre and GlaxoSmithKline Biologicals.

The seroepidemiology of Haemophilus influenzae type b prior to introduction of an immunization programme in Kathmandu, Nepal.

Haemophilus influenzae type b (Hib) is now recognized as an important pathogen in Asia. To evaluate disease susceptibility, and as a marker of Hib transmission before routine immunization was introduced in Kathmandu, 71 participants aged 7 months-77 years were recruited and 15 cord blood samples were collected for analysis of anti-polyribosylribitol phosphate antibody levels by enzyme-linked immunosorbent assay. Only 20% of children under 5 years old had levels considered protective (>0.15 µg/ml), rising to 83% of 15-54 year-olds. Prior to introduction of Hib vaccine in Kathmandu, the majority of young children were susceptible to disease.

Recognising serious umbilical cord anomalies.

Umbilical vessel catheterisation is a common intervention in neonatal care. Many complications are recognised, some of which are life-threatening. We report the case of a term neonate who was compromised at birth following antepartum haemorrhage with evidence of multiorgan ischaemic injury. Following resuscitation and umbilical vessel catheterisation, she developed pneumoperitoneum. At laparotomy, a patent vitellointestinal duct was identified and resected. Intestinal perforation was found in the duct wall, most plausibly explained by the unintentional catheterisation of the duct via the umbilicus. Learning to recognise umbilical cord anomalies, such as patent vitellointestinal duct, can be simple and could prevent potentially serious complications.

Characterisation of murine MICL (CLEC12A) and evidence for an endogenous ligand.

Inhibitory receptors are required for the control of cellular activation and they play essential roles in regulating homeostasis and immunity. We previously identified a human inhibitory C-type lectin-like receptor, MICL (CLEC12A), a heavily glycosylated monomer predominantly expressed on myeloid cells. Here we characterise the murine homolog of MICL (mMICL), and demonstrate that the receptor is structurally and functionally similar to the human orthologue (hMICL), although there are some notable differences. mMICL is expressed as a dimer and is not heavily glycosylated; however, like hMICL, the receptor can recruit inhibitory phosphatases upon activation, and is down-regulated on leukocytes following stimulation with selected TLR agonists. Using novel monoclonal antibodies, we demonstrate that, like the human receptor, mMICL is predominantly expressed by myeloid cells. However, mMICL is also expressed by B cells and CD8+ T cells in peripheral blood, and NK cells in the bone marrow. Finally, we show that mMICL recognises an endogenous ligand in a variety of murine tissues, suggesting that the receptor plays a role in homeostasis.

Structure of the fungal beta-glucan-binding immune receptor dectin-1: implications for function.

The murine molecule dectin-1 (known as the beta-glucan receptor in humans) is an immune cell surface receptor implicated in the immunological defense against fungal pathogens. Sequence analysis has indicated that the dectin-1 extracellular domain is a C-type lectin-like domain, and functional studies have established that it binds fungal beta-glucans. We report several dectin-1 crystal structures, including a high-resolution structure and a 2.8 angstroms resolution structure in which a short soaked natural beta-glucan is trapped in the crystal lattice. In vitro characterization of dectin-1 in the presence of its natural ligand indicates higher-order complex formation between dectin-1 and beta-glucans. These combined structural and biophysical data considerably extend the current knowledge of dectin-1 structure and function, and suggest potential mechanisms of defense against fungal pathogens.

Human MICL (CLEC12A) is differentially glycosylated and is down-regulated following cellular activation.

C-type lectins are the most diverse and prevalent lectin family in immunity. Particular interest has recently been attracted by the C-type lectin-like receptors on NK cells, which appear to regulate the activation/inhibitory balance of these cells, controlling cytotoxicity and cytokine production. We previously identified a human C-type lectin-like receptor, closely related to both the beta-glucan receptor and the lectin-like receptor for oxidized-LDL, named MICL (myeloid inhibitory C-type lectin-like receptor), which we had shown using chimeric analysis to function as an inhibitory receptor. Using a novel MICL-specific monoclonal antibody, we show here that human MICL is expressed primarily on myeloid cells, including granulocytes, monocytes, macrophages, and dendritic cells. Although MICL was highly N-glycosylated in primary cells, the level of glycosylation was found to vary between cell types. MICL surface expression was down-regulated during inflammatory/activation conditions in vitro, as well as during an in vivo model of acute inflammation, which we characterize here. This suggests that human MICL may be involved in the control of myeloid cell activation during inflammation.

C-type lectin-like receptors on myeloid cells.

Host defence against pathogens requires the recognition of conserved microbial molecules, or 'pathogen-associated molecular patterns' (PAMPs), by their receptors termed 'pattern recognition receptors' (PRRs), represented most notably by toll-like receptors (TLRs) and C-type lectins. The 'non-classical' C-type lectins (these that lack the residues involved in calcium binding, required for carbohydrate binding) are traditionally thought of as being restricted to natural killer (NK) or T cells, playing important roles in immune surveillance. In recent years, however, a growing number of these receptors have been identified on myeloid cells, both of human and mouse origin. In contrast to their NK counterparts that primarily control cellular activation through recognition of major histocompatibility antigen (MHC) class I and related molecules, the myeloid-expressed receptors appear to have a far more diverse range of functions and ligands, including those of exogenous origin. Some of C-type lectin-like molecules possess activating/inhibitory signalling motifs that trigger downstream signalling events, suggesting the role for these receptors as positive/negative regulators of granulocyte and monocyte functions. With the exception of a few myeloid NK-like lectins, the natural ligands for most of these receptors remain unidentified, making it difficult to define their functions in normal physiological, inflammatory or pathological conditions. Importantly, in some cases, these novel C-type lectin-like lectins, encoded by genes from the same gene cluster, can act as receptor/ligand pairs, additionally contributing to the regulation of myeloid cell functions or their interaction with other (like NK) cell types. However, the relevance and importance of such interactions still needs to be assessed. Although few of the myeloid-expressed C-type lectins have been characterized in detail, we review here each of these receptors and highlight their prospective roles in innate and adaptive immunity.

The human beta-glucan receptor is widely expressed and functionally equivalent to murine Dectin-1 on primary cells.

We identified the C-type-lectin-like receptor, Dectin-1, as the major receptor for fungal beta-glucans on murine macrophages and have demonstrated that it plays a significant role in the cellular response to these carbohydrates. Using two novel, isoform-specific mAb, we show here that human Dectin-1, the beta-glucan receptor (betaGR), is widely expressed and present on all monocyte populations as well as macrophages, DC, neutrophils and eosinophils. This receptor is also expressed on B cells and a subpopulation of T cells, demonstrating that human Dectin-1 is not myeloid restricted. Both major functional betaGR isoforms - betaGR-A and betaGR-B - were expressed by these cell populations in peripheral blood; however, only betaGR-B was significantly expressed on mature monocyte-derived macrophages and immature DC, suggesting cell-specific control of isoform expression. Inflammatory cells, recruited in vivo using a new skin-window technique, demonstrated that Dectin-1 expression was not significantly modulated on macrophages during inflammation, but is decreased on recruited granulocytes. Despite previous reports detailing the involvement of other beta-glucan receptors on mature human macrophages, we have demonstrated that Dectin-1 acted as the major beta-glucan receptor on these cells and contributed to the inflammatory response to these carbohydrates.

Dectin-1 uses novel mechanisms for yeast phagocytosis in macrophages.

The phagocytosis of pathogens is a critical event in host defense, not only for clearance of the invading microorganism, but also for the subsequent immune response. We have examined Dectin-1, a proinflammatory nonopsonic receptor for beta-glucans, and show that it mediates the internalization of beta-glucan-bearing ligands, including yeast particles. Although requiring tyrosine phosphorylation and the cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM)-like motif, uptake mediated by Dectin-1 was different from any previously reported phagocytic receptor and was not dependent on Syk-kinase in macrophages. Furthermore, intracellular trafficking of this receptor was influenced by the nature of the beta-glucan ligand, which has significance for the biologic activity of these immunomodulatory carbohydrates.

Commentary: C-type lectins on the macrophage cell surface--recent findings.

Interest in the C-type lectin family as a fundamental mediator of diverse immune interactions has grown exponentially in recent years, especially concerning members expressed on NK cells and their roles in anti-viral and anti-tumor immunity. For macrophages, however, collating the roles of the broad range of C-type lectins expressed may be a more complex task. Analysis of the configuration of the C-type lectin(-like) domains among any other conserved modules reveals subfamilies of highly divergent structures. The identified ligands are likewise highly varied; however, most in vivo ligands are unknown and the significance of any recognized interactions is not well understood. In this commentary we review key features of C-type lectin and lectin-like receptor expression on macrophages and discuss recent data that sheds light on their varied functions. We aim to highlight the considerable body of novel/uncharacterized members of this receptor family and to place in context the initial description of CLECSF8, which is reported in this issue of the journal.

Identification and characterization of a novel human myeloid inhibitory C-type lectin-like receptor (MICL) that is predominantly expressed on granulocytes and monocytes.

Inhibitory and activatory C-type lectin-like receptors play an important role in immunity through the regulation of leukocytes. Here, we report the identification and characterization of a novel myeloid inhibitory C-type lectin-like receptor (MICL) whose expression is primarily restricted to granulocytes and monocytes. This receptor, which contains a single C-type lectin-like domain and a cytoplasmic immunoreceptor tyrosine-based inhibitory motif, is related to LOX-1 (lectin-like receptor for oxidized low density lipoprotein-1) and the beta-glucan receptor (Dectin-1) and is variably spliced and highly N-glycosylated. We demonstrate that it preferentially associates with the signaling phosphatases SHP-1 and SHP-2, but not with SHIP. Novel chimeric analyses with a construct combining MICL and the beta-glucan receptor show that MICL can inhibit cellular activation through its cytoplasmic immunoreceptor tyrosine-based inhibitory motif. These data suggest that MICL is a negative regulator of granulocyte and monocyte function.

Dectin-1 mediates the biological effects of beta-glucans.

The ability of fungal-derived beta-glucan particles to induce leukocyte activation and the production of inflammatory mediators, such as tumor necrosis factor (TNF)-alpha, is a well characterized phenomenon. Although efforts have been made to understand how these carbohydrate polymers exert their immunomodulatory effects, the receptors involved in generating these responses are unknown. Here we show that Dectin-1 mediates the production of TNF-alpha in response to zymosan and live fungal pathogens, an activity that occurs at the cell surface and requires the cytoplasmic tail and immunoreceptor tyrosine activation motif of Dectin-1 as well as Toll-like receptor (TLR)-2 and Myd88. This is the first demonstration that the inflammatory response to pathogens requires recognition by a specific receptor in addition to the TLRs. Furthermore, these studies implicate Dectin-1 in the production of TNF-alpha in response to fungi, a critical step required for the successful control of these pathogens.