Observational, longitudinal, prospective, and multicenter real-world evidence (RWE) study. The intervention phase (single arm) lasted 28 days. To obtain data on the use of healthcare resources up to one year of life, an analysis of medical records (hospital visits, infections, medications used) was performed when the individuals reached one year of age. The study was conducted between June 2021 and December 2022 in 10 sites of 3 regions in Argentina. The approval of the Independent Ethics Committee for Trials in Clinical Pharmacology of the Foundation for Pharmacological and Drug Studies (FEFYM) “Luis María Zieher” was obtained. The study was conducted in accordance with Good Clinical Practices and the Declaration of Helsinki. The parents of all recruited patients signed a written informed consent.

Infants with presumptive or confirmed diagnosis of CMPA, who had, as their first indication, an EHF for a minimum period of 28 days. The EHF used in this study was Nutrilon Pepti Syneo® (Nutricia, the Netherlands), a nutritionally complete food for specific medical purposes with hydrolyzed whey protein, long-chain fatty acids and synbiotics (GOSsc/FOSlc and Bifidobacterim breve M-16V), intended for the dietary management of CMPA in infants from birth, either as a sole source of nutrition or as a supplement to breastfeeding and/or complementary foods. The initial evaluation and subsequent follow-up of each participant was performed according to the usual practices for patients with suspected CMPA.

Patients with presumptive or confirmed diagnosis of CMPA who were less than or equal to 8 months of age, with parents already feeding their child with formula, were included. Infants who had already been previously fed hypoallergenic formulas, who suffered from severe CMPA thus requiring an AAF, or who had contraindications for consuming synbiotics (short bowel, parenteral nutrition, post pyloric feeding, central venous catheter and/or immunocompromised infants) were excluded. For follow-up at one year of life, all infants who had completed the 28 days of initial treatment were included. Children who subsequently required an AAF and those for whom data could not be extracted from medical records were excluded from this analysis.

For data collection, Infant Gastrointestinal Symptoms Questionnaire (IGSQ-13) (14) and Cow's Milk Allergy Symptoms Score (CoMiSS) (3) had to be completed by the treating physicians, while Patient Oriented Scoring Atopic Dermatitis (PO-SCORAD) (15) and Family Quality of Life—Parental Burden questionnaire (FAQL-PB) (16) had to be completed by the patient's families.

At baseline, 96.8% of the infants presented gastrointestinal symptoms (60% along with dermatological symptoms, 16.9% with respiratory symptoms, 9.2% with both dermatological and respiratory symptoms, and 10.7% presenting exclusively gastrointestinal symptoms). The remaining 3.2% presented dermatological and respiratory symptoms.

The progression of these symptoms after 4 weeks of treatment with an EHF with synbiotics is shown in Table 2.

At baseline, 21 patients showed a CoMiSS greater than or equal to 12. At the end of the treatment, no patient exceeded it. Figure 3A shows that the median CoMiSS at study onset was 9, decreasing significantly to 3 with treatment (p = 0.000).

Patients included in the study had mild eczema, judging by a median PO-SCORAD at baseline of 11.5 (Interquartile range 1–23). With the treatment the median PO-SCORAD dropped to 1.0 (Interquartile range 1–6) on day 28 (p = 0.000) (Figure 3B).

Figure 4 shows the changes in infants' stools and regurgitation with treatment, as well as the impact of these modifications on the quality of life of their caregivers. The percentage of infants with more than 5 daily stools decreased throughout treatment between day 1 and day 28 (p < 0.05) (Figure 4A). Regarding consistency, 28% of parents reported watery stools on day 1, decreasing to 10% on day 28 (p = 0.004) (Figure 4B).

Figure 4C shows that on day 1, 43% of patients had 4 or more daily regurgitations. After 28 days of treatment, this percentage dropped to 22% (p = 0.01). Within this group, the percentage of patients who had more than 7 regurgitations per day changed from 32% at baseline to only 7% on day 28 (p = 0.0003). The percentage of patients who no longer had any episodes increased from 11% (day 1) to 31% on day 28 (p = 0.003).

Consistent with the above data, 34% of parents reported that their children “were always uncomfortable due to regurgitation” at the beginning of treatment, a percentage that dropped to 2% on day 28 (p = 0.000). On the other hand, only 20% reported not being uncomfortable on day 1, while 58% reported that they never felt uncomfortable on day 28 (p = 0.000) (Figure 4D).

Other relevant aspects for the quality of life of infants and their caregivers are those related to sleeping and crying. At baseline, 13% of patients cried more than 3 h per day, while on day 28 that percentage dropped to 3% (p = 0.03). Children who, according to their parents, did not cry due to irritability changed from 31% at baseline to 49% on day 28 (p = 0.03). Likewise, an improvement in the infants' sleeping pattern was appreciated with the treatment (not shown).

Table 3 shows the impact on the family's quality of life that represents an infant with CMPA and how it improves significantly with treatment. At study onset, 56% of the families reported feeling very overwhelmed, a percentage that dropped to 17% after 28 days of treatment (P < 0.05). The small percentage of families that did not feel overwhelmed at study onset (17%) grew to 43% on day 28 (p < 0.05).

The median FAQL-PB score at baseline was 49 (Interquartile range 27–64), dropping to 30 (Interquartile range 18–54) with the treatment on day 28 (p = 0.02).

More than half of the infants (52%) did not require any consultation to the emergency service until one year of life, while only 6 infants (10.17%) required more than 5 consultations. Of these, 2 patients had to be hospitalized for reasons unrelated to their CMPA.

42.3% of the infants did not require pharmacological medication during follow-up. Only 12% (7 patients) required additional drug treatment for their allergic symptoms, 34% used analgesics and/or antipyretics, and 24% required antibiotics.

Less than half of the infants (n = 29; 49%) presented at least one infectious condition: 2 presented gastrointestinal infections, 18 presented respiratory infections, 3 a combination of both, and 6 patients presented other infections.

When establishing a nutritional treatment for CMPA it is also important to demonstrate an appropriate infant growth and to ensure a proper compliance with the treatment.

All the subjects included in this study presented anthropometric measures (height, weight, and head circumference) within the WHO growth chart percentiles for their sex and age. The average anthropometric measures can be seen in Supplementary Table S1.

Considering some issues of daily family management that contribute to treatment compliance, 98.5% of the caregivers found it easy to prepare the formula, and the acceptance by the infants was 69%. According to the appreciation of the caregivers, 66% of the infants seemed to enjoy it (not shown).

The mean age of patients who entered the study with a diagnosis or suspicion of CMPA agrees with international (10) and local (19) reports. It is known that food allergy is a complex immune disorder caused by specific genetic variants in combination with environmental and nutritional exposures (20). In this context, both the high rate of C-section and the low rate of exclusive breastfeeding appreciated in our sample have been shown to interfere with the homeostasis of the intestinal microbiota and to be determinants of dysbiosis (21, 22), which is considered one of the main responsible for the increasing incidence of allergic disorders (23–25). The family history of atopy present in the recruited infants is also a risk factor associated with CMPA. It is known that the existence of a history of atopy in a sibling increases the risk of allergic disease by 25%–30%, in one parent increases it by 20%–40% and in both parents by 40%–60% (26, 27).

More than 95% of the infants showed complete improvement or resolution of CMPA symptoms with treatment, confirming the previously reported efficacy of the formula (10). Although 92% of the gastrointestinal symptoms and 87.5% of the dermatological symptoms evolved favorably, only 63% of the respiratory symptoms showed this positive outcome. From an epidemiological point of view, as the study was conducted during autumn and winter, an increase in respiratory symptoms not associated with CMPA but with seasonal viruses may have been appreciated. This is partially confirmed by the results of nasopharyngeal swabs that showed the presence of respiratory syncytial virus in some infants.

The treatment efficacy is also evidenced by the improvement of both the CoMiSS (captured by treating physicians) and the PO-SCORAD (captured by caregivers). Previous reports have shown similar results in infants with CMPA receiving an EHF with GOS, FOS and Bifidobacterium breve M-16V (10, 28, 29).

A multicenter, double-blind, placebo-controlled study, in which 90 exclusively formula-fed infants with atopic dermatitis were randomly assigned to receive an EHF with or without synbiotics for 12 weeks, showed that both treatments improved atopic dermatitis, as assessed by the SCORAD index. Although no significant differences were observed between the consumption of an EHF with or without synbiotics in the subgroup of patients with non-IgE-mediated atopic dermatitis, the differences were significant in favor of the consumption of EHF with synbiotics in the subgroup of patients with IgE-mediated atopic dermatitis (28).

On the other hand, it has also been reported that the treatment with EHF with synbiotics enhanced the management of infants with non-IgE-mediated CMPA who were already established on EHF without synbiotics, showing a significant improvement in atopic dermatitis in those patients with severe baseline symptoms (PO-SCORAD reduction from 34.7 to 18.2) (10).

Parents or caregivers of infants with food allergies reported a lower quality of life, due to the social and emotional impact they suffer, and referred high levels of anxiety and stress (30). In our study, parents reported an improvement in their quality of life after 28 days of receiving an EHF with synbiotics. This is in line with was previously reported by Hubbard et al., who also showed a significant reduction in the FAQL-PB score between baseline (mean 30.9) and after 4 weeks of treatment with an EHF with synbiotics (mean 22.6) (10).

Non-IgE-mediated CMPA can present a wide range of gastrointestinal manifestations, including vomiting, regurgitation, or diarrhea (31). Although a randomized clinical trial comparing the consumption of an EHF with and without synbiotics showed that stool consistency was significantly softer in the group that consumed the formula with synbiotics without finding significant differences in relation to stool frequency (28), our results showed that a 28-day treatment with an EHF with synbiotics significantly improved stool consistency but also decreased the number of daily regurgitations and stools. These results were in accordance with the perception of caregivers, who reported that the discomfort that these symptoms generated before treatment decreased substantially after treatment, indicating a substantial improvement in both infants and caregivers' quality of life. For the correct interpretation of these results, it is important to consider two aspects: time and feeding. On the one hand, at least partially, the improvement of these symptoms could have occurred just over time and could not be completely attributed to the treatment. On the other hand, both the frequency and consistency of stools also depended on whether the patient is being breastfed or not. In the present study, we were unable to carry out a subgroup analysis to specify whether the decrease in the frequency and consistency of stools is seen differentially in those infants of mothers who feed their children with breast milk and formula or exclusively with formula.

Other aspects that substantially modified the infant's and the family's quality of life were the sleeping pattern and the crying attributed to irritability. There is a close relationship between food allergy and sleep disorders; an association that has increased in recent times (32). Our data indicated an improvement in the sleeping pattern of infants with CMPA after treatment, reflected both in the increase of those patients who reached what caregivers consider a normal pattern and in the lower percentage of patients with nocturnal awakenings.

Regarding inconsolable paroxysmal crying, it can often be a sign of an underlying medical condition and it may be the first manifestation of a food allergy (33). In our study, the positive evolution of CMPA symptoms led to a significant improvement in the irritability of infants, which was reflected in the decrease in the number of hours of crying after treatment and in the decrease in the crying perceived by parents as related to irritability.

Given that the EHF evaluated in the present study has as a distinctive characteristic the addition of synbiotics with potential modulating effect on the immune system (7), and since the management of an infant with CMPA represents a significant burden for the health system in terms of the use of health resources and medical consultations (10), our study evaluated the patient's medical records at one year of life to describe the number of hospital visits, infections and medication used.

In a recent study, hospital visits and prescriptions of medications were collected from hospital medical records and caregivers, during the 6-months before, and 6-months after EHF with synbiotics treatment initiation. In said study, significant reductions in the mean number of overall hospital visits required by infants were observed in the 6 months after the treatment with EHF with synbiotics. The authors also showed a significant decrease in prescriptions for gastrointestinal conditions (46.2% vs. 7.7% before and after EHF with synbiotics, respectively) (10) which impresses to be in line with our report where only 6.9% of the patients suffered from gastrointestinal infections up to one year of life. Taking these results into account, the low use of healthcare resources observed in our study could also be due to an improvement in the species balance of the intestinal microbiota of patients who received synbiotics which has been previously described (10). It is worth noting that this is only a hypothesis as our study, being a RWE study and not a randomized controlled trial, lacks a control group.

Despite this important limitation, the data obtained in our study together with contributions such as those of Hubbard et al. (10) can help to understand the role of synbiotics in the management of CMPA through changes in the composition of their intestinal microbiota that bring it closer to that of a healthy child.

Although the study had some palatability-related dropouts, a high percentage of infants entering the study protocol appeared to enjoy consuming the formula, as reported by their caregivers. This better palatability of the EHF used in this study may be since it contains whey proteins and lactose, which would result in better palatability than formulas with casein and without lactose (34).

Both an easy preparation and acceptance of an EHF by infants have been reported in the present study and are essential to achieve a good adherence to treatment (35).

The main strength of the study was to be one of the first reports to evaluate the progression of CMPA symptoms treated with an EHF with synbiotics, in a real world setting with a significant sample.

One of the limitations of this study was the relatively high dropout rate, mainly for three reasons: change of treating physician, palatability, or AAF requirement. In relation to palatability, it is important to consider that, since one of the exclusion criteria for the study was having previously consumed hypoallergenic formulas, all the children who entered the protocol had been exclusively breastfed or consuming infant formulas, both more palatable options than an EHF. On the other hand, since those patients who required the consumption of AAF were excluded from the analysis (selective dropout), the results of the study may be slightly affected by attrition bias. Another limitation of the study is that the initial diagnosis of CMPA was based on the clinical judgment of the treating physician, without necessarily being confirmed by a diagnostic oral food challenge. Finally, other weaknesses of the study are that, as being a RWE study, lacks a control group and that its results cannot be extrapolated to other types of synbiotic combinations, as the results are specific for the GOS/FOS and Bifidobacterium breve M-16V combination.