After a long break over Christmas, New Year and several periods of clinical service, I have a few posts that I am developing, hopefully you will all find them useful.
Lactoferrin is a molecule present in mammalian milk which acts as an iron transporter, hence the name. It binds to species specific receptors on enterocytes and permits iron absorption, then being recycled into the intestinal lumen to promote more iron absorption. For some reason mammals evolved to produce a molecule that also has multiple antimicrobial activities, anti-bacterial, -viral -fungal.
Lactoferrin is the principle component of whey in human milk, and a very similar protein in bovine milk is present in much lower concentrations, but has more active antimicrobial properties. As very preterm babies often have very little breast milk intake in the first few days of life they often receive very little lactoferrin.
A very well-performed multicenter trial in Italy, published a few years ago now, showed a dramatic reduction in late-onset sepsis among infants randomized to either bovine lactoferrin (100 mg per day regardless of weight), or bovine lactoferrin plus a strain of lactobacillus rhamnosus (a probiotic organism), compared to placebo. (Manzoni P, et al. Bovine lactoferrin supplementation for prevention of late-onset sepsis in very low-birth-weight neonates: a randomized trial. JAMA. 2009;302(13):1421-8). Among just over 150 babies per group (n=470 total), lactoferrin reduced the proportion of babies with at least one episode of culture-positive late-onset sepsis from about 17% to about 5% in the 2 lactoferrin groups. In that study the average birthweight of the infants was about 1100g, their average (mean) gestational age was about 29.5 weeks and there were 168 babies under 1 kg. When scrutinizing the results closely, there were a few more fungal sepsis episodes than some other groups have reported, of the 45 total babies with sepsis, 11 had a fungal infection, 9 of them among controls; and there were fewer CoNS (coagulase negative staphylocci) than some other studies, only 4 of the 45 episodes were CoNS. This was partly because of requiring 2 positive cultures for a CoNS diagnosis, if you add those with only 1 positive culture another 9 patients were positive with CoNS infection, and I don’t know what the total number of infected babies would then become, but the proportion of bacterial sepsis that is CoNS then becomes similar to other reports.
Why am I concentrating on this older study, you may ask? It is because the ELFIN trial has just been published (in the Lancet Griffiths J, et al. Enteral lactoferrin supplementation for very preterm infants: a randomised placebo-controlled trial. The Lancet. 2019;393(10170):423-33) open access and a complete document in “Health Technology Assessment” (Griffiths J, et al. Enteral lactoferrin to prevent infection for very preterm infants: the ELFIN RCT. Health technology assessment. 2018;22(74):1-60), which is a freely available download.
ELFIN recruited 2200 babies less than 32 weeks gestation at less than 72 hours of age, to receive lactoferrin (150 mg/kg/day; max of 300 mg/day) or placebo, mixed with a milk feed. The primary outcome was at least one late-onset infection, suspected or proven. Proven sepsis required a positive culture as well as clinical signs requiring a blood culture and an intention to treat for 5 days at least, suspected infection was defined as follows:
Absence of positive microbiological culture, or culture of a mixed microbial flora or of probable skin contaminants (ie, diphtheroids, micrococci, propionibacteria) only, and treatment or clinician intention to treat for 5 days or more with intravenous antibiotics (excluding antimicrobial prophylaxis) after the investigation was undertaken for an infant who presents at least 3 of the following clinical or laboratory features of invasive infection:
•increase in oxygen requirement or ventilatory support
•increase in frequency of episodes of bradycardia or apnoea
•ileus or enteral feeds intolerance or abdominal distention
•reduced urine output to less than 1 mL/kg per h
•impaired peripheral perfusion (capillary refill time longer than 3 seconds, skin mottling or core-peripheral temperature gap greater than 2°C)
•hypotension (clinician-defined as needing volume or inotrope support)
•irritability, lethargy, or hypotonia (clinician-defined)
•increase in serum C-reactive protein concentrations to more than 15 mg/L or in procalcitonin concentrations to 2 ng/mL or more
•white blood cells count smaller than 4 × 109/L or greater than 20 × 109/L
•platelet count less than 100 × 109/L
•glucose intolerance (blood glucose smaller than 40 mg/dL or greater than 180 mg/dL)
•metabolic acidosis (base excess less than −10 mmol/L or lactate concentration greater than 2 mmol/L)
We can always debate the relevance of culture negative sepsis, but these criteria are as reasonable as any others (or more-so), and the results for solely culture-positive sepsis are presented as well, as they were an important secondary outcome.
There were 650 episodes of sepsis (confirmed and suspected) and 370 of culture positive, confirmed, sepsis. Episodes were evenly divided between groups, in other words there was no evident impact of the intervention on sepsis. The adjusted risk ratio was 0.95 with lactoferrin, and 95% confidence intervals include a 14% reduction or a 4% increase in all sepsis. Confirmed infection was 17% in each group, RR 1.05, 95% CI 0.87-1.26.
About 2/3 of the confirmed cases were CoNS, there were only 5 total invasive fungal infections, and there was no evidence of any adverse effect of the lactoferrin.
How does this study compare to Manzoni et al? Why might the results be so different?
The control group rate of culture-positive late-onset sepsis is actually quite similar in the two trials at close to 17%, despite the Italian study having slightly smaller infants (they were all < 1500 g whereas 18% of the ELFIN babies were > 1500 g), the gestational ages were very similar. Of course this discounts the infants in the Italian study that only had one culture positive for CoNS (in ELFIN a single culture was sufficient for those infants), so if you add babies who would be considered positive in other studies the proportion of babies with at least one septic episode in the Italian study was even higher. I find this sepsis rate somewhat on the high side for both studies, in the Canadian Neonatal Network the proportion of babies with at least one infection under 32 weeks is less than 10%. (The data available are actually under 31 weeks (about 11%) or under 33 weeks (about 8.5%). The 2 groups of infants 29-30 weeks and 31-32 weeks both had a 3% incidence of sepsis in 2017. The distribution of organisms is somewhat different between the studies, but I don’t think that is enough to explain the differences, if lactoferrin was extremely good at preventing fungal sepsis, and had little effect on other bacterial infections, that might partly explain the differences in results, but only partly. And in fact if you look at the details of the organisms in the report of the Manzoni trial there seemed to be a substantial reduction in gram negative and gram positive bacterial infections, as well as fungal.
Perhaps the preparation of the lactoferrin might have had an impact on its efficacy, the molecule is large and somewhat unstable, impacts on its concentration and efficacy have been noted according to how it is prepared. Heat treatment of stored breast milk markedly reduces lactoferrin concentrations for example, (Li Y, et al. Pasteurization Procedures for Donor Human Milk Affect Body Growth, Intestinal Structure, and Resistance against Bacterial Infections in Preterm Pigs. J Nutr. 2017;147(6):1121-30. Schwarcz WD, et al. Conformational changes in bovine lactoferrin induced by slow or fast temperature increases. Biological chemistry. 2008;389(8):1137-42) and the way it is dried affects the composition of the resulting powder (Wang B, et al. Characteristics of bovine lactoferrin powders produced through spray and freeze drying processes. Int J Biol Macromol. 2017;95:985-94).
Perhaps feeding practices have changed enough that the control ELFIN babies were receiving enough fresh colostrum/maternal breast milk in the first few days of life that there were smaller differences in lactoferrin intakes between groups. Manzoni’s babies started feeds on average between 2 and 3 days of age, increased feeds by 10 mL per day and achieved full feeding by the 12th to 14th day of life. There is no mention of donor milk, the alternative to maternal breast milk appears to have been formula, and preterm formulae contain almost no lactoferrin. The data available from the ELFIN trial don’t have much detail, but about 4% of the infants received formula alone, just less than 70% received mixed feeding, and the remainder exclusively breast milk. A subgroup analysis of ELFIN showed a potential effect of lactoferrin among babies receiving mixed feeds.
As you can see the p-value for the interaction, lactoferrin and type of milk, was not significant, so we need to be very careful making any sort of conclusion, but it is at least possible that there is an impact of lactoferrin supplementation among infants receiving less lactoferrin from breast milk.
Another explanation is that there are biases introduced into either one or other of the trials that had the effect of lactoferrin seeming ineffective in ELFIN, and highly effective in the Manzoni trial, I can’t imagine what those biases may be, but the only other explanation of there being such a difference between ELFIN and the Manzoni trial is random variation, and that the Manzoni trial, a prospective masked multi-center randomized trial with reasonable sample size, was the 1:1000 occurrence of a p-value of about 0.001. I find that explanation extremely unsatisfying.
What should we do now? I think the first priority is to finish any other trials that are in progress, obtain as much information as possible about what seemed to be such a promising intervention. I think that can ethically be justified as there is no trace of adverse effects of lactoferrin in any of the trials to date, it is an intervention with some evidence of efficacy, quite inexpensive, and with a physiologic rationale. Even with ELFIN the results of the trials currently available do leave the possibility of a substantial benefit of lactoferrin supplementation, although the 95% confidence intervals probably do not approach the impact shown by Manzoni. Once all the data are in, a collaborative examination of the data to try to identify the reasons behind these discrepancies, and perhaps design future trials of the most promising product, in the most promising groups of babies.