Late-onset infection in very preterm infants

Late-onset invasive infection (occurring >72 h after birth) is the most common serious complication associated with intensive care for newborn infants. The incidence is >20% in very preterm or very low birth weight infants reflecting their level and duration of exposure to invasive procedures and intensive care.1–3 Coagulase-negative staphylococci cause about half of all bloodstream infections.4 Other pathogens include Gram-negative bacilli (mainly enteric bacilli), Gram-positive cocci (Staphylococcus aureus, enterococci), and fungi (predominantly Candida spp.).3 ,5 ,6

Very preterm infants with late-onset invasive infection have a higher risk of mortality and a range of important morbidities including necrotising enterocolitis (NEC), retinopathy of prematurity (ROP), bronchopulmonary dysplasia (BPD) and the need for intensive care and mechanical ventilation.1 ,7 ,8 These higher rates of mortality and serious morbidity are usually associated with Gram-negative bacterial or fungal infection. Coagulase-negative staphylococcal infection, although common, is generally associated with a more benign clinical course. However, even ‘low grade’ coagulase-negative staphylococcal bloodstream infection may generate inflammatory cascades associated with both acute morbidity (metabolic, respiratory or thermal instability) and long-term white matter and other brain damage that may result in neurodevelopmental disability.9 As a consequence of these associated morbidities, very preterm infants with invasive infection spend about 20 more days in hospital than their peers without infection.2 Late-onset infection therefore has major consequences for perinatal health care and service management, delivery and costs.

Diagnosis of late-onset invasive infection

Clinical signs of invasive infection in very preterm infants individually have limited sensitivity. Similarly, laboratory measures (biomarkers) are insensitive and have low predictive value for bloodstream infection.10 Most neonates who undergo ‘sepsis evaluation’ and are treated empirically with antibiotics do not have infection confirmed subsequently. Since individual clinical signs and laboratory markers are generally unreliable predictors of true invasive infection, a policy of early empirical treatment when infection is suspected inevitably results in unnecessary exposure to antibiotics. This practice results in more interventions for very preterm infants and may drive the emergence of antibiotic-resistant pathogens including vancomycin-resistant enterococci, methicillin-resistant S aureus and fluconazole-resistant fungi in neonatal care centres.11

Infection control measures such as hand-washing and intravascular catheter care ‘bundles’ (as well as interventions to promote the use of maternal breast milk rather than formula) can help prevent late-onset invasive infection, but benchmarking and quality improvement studies in neonatal networks have indicated that infection rates plateau following adoption of these practices.12–14 Given the burden of mortality, acute and long term morbidity, and costs to families and health services, there is a need to develop and assess novel and innovative strategies to prevent late-onset invasive infection in very preterm infants. One such promising intervention is enteral supplementation with the processed cow milk protein lactoferrin.

Lactoferrin

Lactoferrin, a member of the transferrin family of iron-binding glycoproteins, is a key component of the mammalian innate response to infection. Lactoferrin is the major whey protein in human colostrum (∼6 mg/ml) and breast milk (∼1 mg/ml, comprising ∼7% of total breast milk protein) and is also present in tears, saliva, cerebrospinal fluid and other secretions. Lactoferrin has broad microbicidal activity (box 1).15 ,16

Development of resistance to lactoferrin would require multiple simultaneous mutations, perhaps explaining why lactoferrin remains a potent inhibitor of viruses, Gram-positive cocci, Gram-negative bacilli and fungi including Candida spp., after millions of years of mammalian evolution.15–18

Lactoferrin also has prebiotic properties, creating an enteric environment for the growth of beneficial bacteria and reducing colonisation with pathogenic species, and has direct intestinal immunomodulatory and anti-inflammatory actions by affecting cytokine expression, mobilising leucocytes into the circulation and activating T-lymphocytes.19–22 Lactoferrin undergoes partial acid proteolysis in the stomach to form lactoferricins, peptides with enhanced antimicrobial activity.23 This may explain partly the link between exposure to H2-receptor antagonists and the risk of late-onset invasive infection in very preterm infants.24

At high concentration, as in colostrum, lactoferrin enhances proliferation of enterocytes and closure of enteric gap junctions. At lower concentrations, lactoferrin stimulates differentiation of enterocytes and expression of intestinal digestive enzymes.25 Lactoferrin suppresses free radical activity when iron is added to milk, suggesting that it may have further anti-inflammatory actions that could modulate the pathogenesis of diseases linked with free radical generation such as NEC, ROP and BPD.26

Enteral lactoferrin immunoprophylaxis

Very preterm infants have low lactoferrin intake exacerbated by the delay in establishing enteral feeding.27 Enteral lactoferrin supplementation may therefore compensate for this gestational immunodeficiency. To date, most research effort has focussed on assessing the prophylactic effects of bovine lactoferrin, which is greater than 70% homologous with human lactoferrin but has higher antimicrobial activity, is inexpensive, and is available commercially as a food supplement in a stable powder form. Bovine lactoferrin has been a component of the human infant diet for thousands of years and is registered as ‘Generally Recognised As Safe’ by the US Federal Drug Administration with no reports of human toxicity. The ‘no-observed-adverse-effect level’ of bovine lactoferrin is greater than 2 g/kg/day in rodents.28 Since the affinity of bovine lactoferrin for the human small intestine lactoferrin receptor is low, absorption of intact bovine lactoferrin is negligible.29 It is considered therefore that the allergenic risk is unlikely to be increased significantly by the administration of bovine lactoferrin.30

The only randomised controlled trial of prophylactic lactoferrin supplementation in very preterm infants was conducted in 11 Italian neonatal centres in 2007 and 2008.31 In total, 472 infants participated in the three-arm trial. The investigators reported that enteral supplementation with either lactoferrin or with lactoferrin plus the probiotic Lactobacillus rhamnosus GG (LGG) reduced the incidence of late-onset invasive infection by two thirds compared with controls (table 1). The incidence of NEC was decreased in the lactoferrin plus LGG group only (0% vs 6% in controls). The effect size was similar whether infants were fed predominantly with human or formula milk.

The incidence of severe ROP was also lower in the lactoferrin group (3.9% vs 11.3% in controls). The trial did not find any evidence of an effect on the incidence of severe intraventricular haemorrhage, BPD, receipt of surgical ligation for patent ductus arteriosus, or all-cause mortality prior to hospital discharge. Longer term neurodevelopmental outcomes have not yet been reported.

Is this existing evidence sufficient to justify a change in practice?

The Cochrane review concludes that available evidence is insufficient to support a change in practice and that the effect of lactoferrin supplementation in preventing late-onset invasive infection and major morbidity and mortality in very preterm infants ‘needs to be confirmed in well-designed, adequately-powered, multicentre (randomised controlled trials)’.32

These cautious conclusions can be justified for several reasons. The effect on reducing invasive infection rates detected in the Italian trial was strongest for the extremely low birth weight (ELBW: <1000 g) subgroup of infants. A statistically significant effect was not found for infants weighing 1000–1500 g. This is consistent with a lower incidence of invasive infection in larger infants and a diminishing dose-response effect reflecting the fixed dosage of 100 mg/day. ELBW infants received lactoferrin for 6 weeks compared with 4 weeks for infants weighing 1000–1500 g. It is plausible that lactoferrin supplementation may have additional beneficial effects if given at daily doses closer to levels ingested by enterally-fed term infants (100–200 mg/kg/day) and for all infants until they reach a post-menstrual age of 34 weeks to cover the periods of highest exposure to infection risk.3 Determining effectiveness (and cost-effectiveness to allow comparison with other infection control interventions) in this population is essential to justify the prophylactic use of this intervention in all very preterm infants.

Although the Italian setting of the existing trial is broadly similar to other high-income settings, there are important differences in clinical practice, particularly in infection control policies and in antibiotic prescribing and stewardship that limit generalisability. The incidence of invasive fungal infection in the control group of participants in the Italian trial was 5.4% overall and 10% in the ELBW subgroup, five-fold higher than the average incidence in UK neonatal units.6 In the Italian trial, a substantial proportion of the overall effect on reducing late-onset invasive infection was due to the effect on reducing fungal infection and there was not a statistically significant effect on the incidence of bacterial infection.33 If the Italian trial data are an over-estimation of effect size, we may be exposing all infants to an intervention with a much higher number needed to treat for benefit than anticipated. This has logistical consequences and opportunity costs that need to be considered.

The ELFIN trial

The pre-clinical data, studies using animal models, and the results of the Italian trial have generated considerable interest and enthusiasm for further trials to assess lactoferrin immunoprophylaxis in very preterm infants. Several international groups are planning large trials. In the UK, the ‘Enteral LactoFerrin In Neonates’ (ELFIN) Trial Group (a collaboration of service-user representatives, clinicians, and trial unit experts) is undertaking a simple and pragmatic randomised controlled trial in which 2200 very preterm infants will be able to participate. Late-onset invasive infection is the pre-specified primary outcome.34 The trial will also be powered to assess meaningful effects on mortality, major morbidity, antibiotic usage and duration of hospital stay. The trial data will contribute to a prospective meta-analysis collaboration with similar trials planned in Australasia and North America. An economic evaluation will be conducted to assess whether the intervention is likely to be cost-effective.