Objective To determine whether the probiotic Lactobacillus reuteri DSM 17938 reduces crying or fussing in a broad community based sample of breastfed infants and formula fed infants with colic aged less than 3 months.
Design Double blind, placebo controlled randomised trial.
Setting Community based sample (primary and secondary level care centres) in Melbourne, Australia.
Participants 167 breastfed infants or formula fed infants aged less than 3 months meeting Wessel’s criteria for crying or fussing: 85 were randomised to receive probiotic and 82 to receive placebo.
Interventions Oral daily L reuteri (1×108 colony forming units) versus placebo for one month.
Main outcomes measures The primary outcome was daily duration of cry or fuss at 1 month. Secondary outcomes were duration of cry or fuss; number of cry or fuss episodes; sleep duration of infant at 7, 14, and 21 days, and 1 and 6 months; maternal mental health (Edinburgh postnatal depression subscale); family functioning (paediatric quality of life inventory), parent quality adjusted life years (assessment of quality of life) at 1 and 6 months; infant functioning (paediatric quality of life inventory) at 6 months; infant faecal microbiota (microbial diversity, colonisation with Escherichia coli), and calprotectin levels at 1 month. In intention to treat analyses the two groups were compared using regression models adjusted for potential confounders.
Results Of 167 infants randomised from August 2011 to August 2012, 127 (76%) were retained to primary outcome; of these, a subset was analysed for faecal microbial diversity, E coli colonisation, and calprotectin levels. Adherence was high. Mean daily cry or fuss time fell steadily in both groups. At 1 month, the probiotic group cried or fussed 49 minutes more than the placebo group (95% confidence interval 8 to 90 minutes, P=0.02); this mainly reflected more fussing, especially for formula fed infants. The groups were similar on all secondary outcomes. No study related adverse events occurred.
ConclusionsL reuteri DSM 17938 did not benefit a community sample of breastfed infants and formula fed infants with colic. These findings differ from previous smaller trials of selected populations and do not support a general recommendation for the use of probiotics to treat colic in infants.
In this large community cohort of infants with colic sufficiently severe for parents to seek assistance from an emergency care setting, treatment with Lactobacillus reuteri did not reduce crying or fussing, nor was it effective in improving infant sleep, maternal mental health, family or infant functioning, or quality of life. In fact, contrary to the hypothesis, infants in the L reuteri group fussed more than those in the placebo group at all post-intervention time points except 6 months. This increased fussing occurred only in formula fed infants; L reuteri did not affect crying or fussing time in exclusively breastfed infants. L reuteri treatment did not lead to changes in infant faecal microbial diversity,Escherichia coli colonisation, or calprotectin levels.
Strengths and limitations of this study
This is the largest randomised controlled trial of probiotic intervention in infants with colic to date. It is the first to include formula fed infants and to extend outcomes to infant sleep, maternal mental health, and family functioning. We used rigorous methods and high quality measures; in particular, the well validated Baby Day Diary reduces carer opportunity for recall bias and is sufficiently detailed to permit separated analyses of infant crying versus fussing. Retention and reported adherence rates were high, with the final sample size adequate to achieve the predetermined statistical power and significance.
The trial has some limitations. Most of the infants were recruited from an emergency or urgent care setting; whereas our results may not therefore generalise to infants whose carers do not seek assistance, treatment in this group may be less relevant. Funding permitted completion of laboratory analyses of a subset of faecal samples collected at 1 month only. The low colonisation rate of L reuteri in only 45% of the treatment infants may reflect low sensitivity of the assay used. It could also reflect low adherence, although we ascertained good adherence by parental report, and equivalent adherence between the two groups by weights of study bottles before and after ingestion of study product. It remains possible that the infants did not receive a dose of L reuteri large enough to establish an effect; however, the trial’s probiotic dosing was the same as that of three previous trials reported as effective.32 33 36 Moreover, the rate of colonisation of L reuteri in normal healthy infants given the probiotic is unknown and possibly highly variable. We excluded infants with a likely diagnosis of allergy to cow’s milk protein, so our results cannot generalise to this subset of infants.
Interpretation in light of other studies
Despite using the same probiotic strain, formulation, and dosing, our negative trial results contrast with the three previous trials of L reuteri in infants with colic,32 33 36 even when considering only breastfed infants. Our trial also failed to demonstrate positive probiotic effects in formula fed infants with colic. There are several possible reasons for the differences in results. Firstly, our sample size was larger than any previous trial and was adequately blinded, unlike Savino and colleagues’ trial.32 Secondly, ours was the only trial that used a validated diary to measure the primary cry or fuss outcome, reducing significantly the likelihood of recall bias and enabling us to uniquely separate out crying and fussing outcomes. Thirdly, the baseline gut microbiota of infants from Melbourne, Australia, could differ from that of Polish and Italian infants, or could be less susceptible to the effects of probiotics. Previous studies have shown that infants with allergic disease have different gut microbiota according to different geographical locations, even among countries with westernised lifestyles.44 45 46 47 48 Fourthly, 60% of our infants had a family history of atopy, in keeping with local epidemiology.49However, the prevalence of a family history of atopy was balanced between groups, and subgroup analysis of infants with and without an atopic family history was similar. Fifthly, it may be that L reuteri in formula fed infants is only effective if infants are also receiving a dairy-free diet. Finally, we included infants who were receiving proton pump inhibitors at the start of the trial. However, proton pump inhibitors are ineffective in reducing crying in irritable infants50 51 and therefore should have no impact on results.
Unexpectedly, infants in the probiotic group fussed significantly more than infants in the placebo group at all time points from day 7 to 1 month in the adjusted analyses. Further analysis revealed that carers from the placebo group had 40 to 60 minutes/day more contact (carrying/moving) with their infants at all time points, although these differences never reached significance (all P>0.05). The relevance of this is uncertain, as increased contact with carers has been shown to reduce fussing and crying in infants without colic52 53 but not in infants with colic.54 Previous studies have shown that in exclusively breastfed infants the use of hypoallergenic formula or dairy elimination diets in mothers is somewhat effective in reducing irritability in infants with colic.10 11 12Interestingly, more infants in the placebo group were receiving hypoallergenic formula at 1 month (eight in placebo group v four in probiotic group, P=0.08), and more exclusively breastfed infants in the placebo group had mothers who were on dairy elimination diets at baseline (table 2) and at 1 month (10 in placebo group v 3 in probiotic group, P=0.03). However, these numbers were all small, and in further analyses the results were not altered by controlling for dairy exclusion and use of hypoallergenic formulas.
In our preliminary analysis of faecal samples, calprotectin levels, although unrelated to trial arm, were lower in those with resolved colic. This finding is in keeping with a previous small study showing faecal calprotectin levels to be higher in infants with colic than in those without,8 but contrary to a larger study showing no differences in faecal calprotectin levels between infants with and without colic.9 Considering that faecal calprotectin levels are associated with intestinal neutrophilic inflammation, it is conceivable that the more than 50% reduction in crying or fussing may reflect lower levels of intestinal inflammation by a mechanism unrelated to probiotic ingestion. These can be further explored by better designed studies investigating faecal calprotectin levels and infant crying.
When the results of our trial are added to the meta-analysis of previous randomised controlled trials of L reuteri for the management of infant colic,31 the overall effectiveness of the probiotic is diminished: mean difference 48 minutes/day (95% confidence interval -84.8 to −11.3) compared with 68 minutes/day (−99.8 to −35.6); figures 3⇓ and 4⇓). However, it should be noted that the meta-analysis puts significant weighting on the study by Savino and colleagues,32 which was an unblinded, open label trial.
Conclusions and future directions
Based on the null findings from this large, rigorous trial, and the results from the updated meta-analysis, which has potential biases, we recommend caution in formulating recommendations regarding use of L reuteri DSM 17938 for reducing crying or fussing in breastfed, and particularly in formula fed, infants with colic. Currently, there are at least five other similar randomised controlled trials taking place in different parts of the world.55 It will be valuable to pool data from all existing and ongoing rigorous trials into an individual patient level meta-analysis to investigate with greater certainty which subgroups of infants with colic, if any, could benefit from probiotics.
What is already known on this topic
Previous small trials suggest that the probiotic Lactobacillus reuteri effectively treats colic in breastfed infants
These studies, however, had limitations as they examined a highly selective group of infants with colic
The effects of L reuteri on formula fed infants with colic are unknown
What this study adds
L reuteri treatment did not reduce crying or fussing in infants with colic, nor was it effective in improving infant sleep, maternal mental health, family or infant functioning, or quality of life
Probiotics therefore cannot be routinely recommended for all infants with colic
Further research is needed to identify which subgroups of infants with colic may benefit from probiotics