Effect of probiotics on C-reactive protein levels in preterm infants: Secondary analysis of a randomized controlled trial

Abstract

BACKGROUND:

Excessive inflammation is associated with adverse outcomes in preterm infants. C- reactive protein (CRP) is a marker of inflammation/infection. Probiotics have anti-inflammatory properties. Randomized controlled trials (RCTs) in preterm infants have not reported effect of probiotics on CRP.

AIM:

To evaluate effect of probiotics on CRP in preterm infants who had participated in a RCT of Bifidobacterium breve (B. breve) m-16v.

METHODS:

Data on all infants (GA <33 weeks, n = 159) enrolled in the RCT was analyzed. For study purpose, CRP <15 mg/L and ≤10 mg/L was considered normal for the first week, and thereafter respectively. Mixed logistic regression modelling was used to assess probiotic effect on CRP levels.

RESULTS:

There were 1579 CRP measurements (Probiotic: 851 vs. Placebo: 728). Baseline characteristics and number [Median (IQR)] of CRP estimations per infant [l0 (5, 20) vs. 10 (6, 17), p = 0.861] were comparable between probiotic vs. placebo group. There was no significant difference in the proportion of infants with high CRP over time (treatment by weekly time points interaction, p = 0.187), and across all time points between probiotic and placebo group (adjusted OR: 1.62, 95% CI: 0.91–2.88, p = 0.102)

CONCLUSION:

B. breve m-16v did not decrease CRP levels in preterm infants born <33 weeks.

Keywords

C-reactive protein inflammation preterm infant probiotic

1 Introduction

Inflammation is increasingly being recognized as a critical contributor to neurological injury in preterm infants [1, 2]. The commonest lesion associated with inflammation in the preterm infant is white matter injury (WMI), which is characterized by focal cystic periventricular leukomalacia and/or diffuse necrosis. It is well known that even low level inflammation can result in lower cognitive, language, and motor development and slower gait velocity in very preterm infants [3].

Late onset sepsis (LOS) and necrotizing enterocolitis (NEC ≥Stage II) are the two commonest conditions associated with inflammation in preterm very low birth weight (VLBW) infants [4, 5]. The most commonly used inflammatory marker to diagnose and monitor the progress in neonatal sepsis and NEC is CRP [6]. CRP is a 115,000-dalton cyclic pentameric protein made of five protomers, each consisting of 206 amino acids [7]. CRP is predominantly secreted by the liver and its production is mainly controlled by IL–6 and to lesser extent IL-1β [8]. Tumor necrosis factor α (TNF-α) and IL-1β are also regulatory mediators of CRP synthesis [9].

Probiotics are live microorganisms that when administered in adequate amounts, exert a beneficial effect on the host [10]. Probiotics have been shown to reduce the risk of ≥ Stage II NEC, mortality, LOS, and the decrease the time taken to achieve full enteral feeds in preterm VLBW neonates [11 –13]. Probiotic strains such as Lactobacillus reuteri and Bifidobacterium bifidum have been shown to decrease proinflammatory cytokines (e.g. IL-6, IL-10, TNF-alpha) and activate the production of anti-inflammatory cytokines [14 –17].

Between November 2010 and May 2012, we conducted a double-blind randomized controlled trial (RCT) to independently assess the product quality, and confirm that Bifidobacterium breve (B. breve) m-16v supplementation will increase fecal B. breve counts [18]. It enrolled a total of 159 preterm neonates <33 weeks (Probiotic: 79, Placebo: 80). Given the availability of data from this RCT, we aimed to assess the anti-inflammatory effect of B. breve m-16 v in the infants enrolled in our trial. We hypothesized that infants enrolled in the B. breve m-16 v arm will have lower CRP values during the period of probiotic supplementation compared to those in the placebo arm.

2 Methods

2.1 Design

This exploratory study involved secondary analysis of data from preterm VLBW infants enrolled in the previously published probiotic (PANTS) trial that assessed B. breve m-16 v against placebo (Maltodextrin) [18]. Preterm VLBW (Gestation: ≤32⁺⁶ weeks and Birth weight: <1500 g) infants ready to commence or on enteral feeds for <12 hours, were eligible for enrolment. The primary outcome was the effect of B. breve m-16v supplementation on levels of B. breve in the stools of preterm neonates as detected by quantitative PCR. Secondary outcomes included evidence of a bifidogenic effect (elevation of total bifidobacteria in stools); incidence of NEC (≥Stage II) and all cause death; time to reach full enteral feeds (150 ml/kg/day) and blood culture positive LOS beyond 72 hours of life. The probiotic dose was 3×10⁹ cfu/day (1.5 ml of the reconstituted solution), given as a single dose via the orogastric feeding tube. For infants ≤27 weeks the daily dose was 1.5×10⁹ cfu per day until reaching milk feeds of 50 ml per kg per day. It was then increased to 3×10⁹ cfu per day. The supplementation was continued until a PMA of 37 weeks or discharge.

2.2 Data on CRP

All infants (n = 159) enrolled in the trial were eligible for inclusion in this post-hoc analysis. The number of measurements and the CRP levels of each infant were recorded from day of admission until discharge/death. The number of infants with CRP measurements in each week, the minimum and maximum CRP and the weekly distribution of peak CRP measurements in each study group were recorded. A total of 8 infants were excluded from analysis: 6 were exclusions from the trial; 2 were exclusions due to no CRP measurements. The study flow diagram is given in Fig. 1.

2.3 Statistical analysis

Fig.1

CONSORT flow diagram.

In the original RCT, sample sizes of 50 per group were estimated to achieve 90% power to detect the colonization rate of 30% in the probiotic versus 5% in the control group when using a two-sided test of proportions with continuity correction at 5% significance level. An additional 20 infants (10 per arm) were enrolled to cover for loss to follow up. Continuous data were summarized with medians, interquartile ranges (IQR) and ranges (R) and categorical data with frequency distributions. Univariate comparisons between probiotic and placebo groups were made using the Mann-Whitney test for continuous outcomes and the Chi-square or Fisher exact test for categorical outcomes. For the first 7 days, CRP ≥15 mg/L was considered high, and from day 8 onwards, CRP >10 mg/L was considered high. During the neonatal period, an upper normal level of CRP is generally accepted as 10 mg/L. However, these values range from 6 to 20 mg/L [19]. Considering that the CRP increases physiologically after birth or due to non-infectious causes (e.g. stressful delivery, perinatal asphyxia, maternal fever during fever, prolonged rupture of membrane), we considered CRP ≥15 mg/L as high during the first week of life [20].

Table 1

Maternal and infant demographic characteristics

	Probiotic N = 77	Placebo N = 76	p-value
Maternal characteristics
Maternal PIH	22 (29%)	15 (20%)	0.202
Maternal APH	20 (26%)	14 (19%)	0.280
Chorioamnionitis	9 (12%)	11 (15%)	0.609
PPROM >24 hours	16 (21%)	23 (31%)	0.178
Maternal antibiotics	65 (84%)	65 (86%)	0.848
Antenatal glucocorticoids
Complete	33 (43%)	39 (51%)	0.530
Incomplete	35 (46%)	28 (37%)
None	9 (12%)	9 (12%)
Inborn	75 (97%)	72 (95%)	0.442
Cesarean section	58 (75%)	49 65%)	0.143
Gestation (weeks)	29 (26–30; 23–32)	28 (26–29; 23–33)	0.622
Gestation ≤27 weeks	28 (36%)	29 (38%)	0.818
Neonatal characteristics
Birth weight	1090 (755–1280;	1025 (810–1260;	0.965
(grams)	466–1830)	480–1770)
Male	45 (58%)	41 (54%)	0.575
SGA	25 (33%)	25 (33%)	0.522
Apgar <7 at 5 minutes	14 (18%)	19 (25%)	0.305
CRIB score	6 (3–9; 1–16)	7 (4–10; 1–16)	0.648
Steroid for CLD	5(6.4%)	6(7.9%)	0.765
PDA	31 (40%)	34 (45%)	0.575
NBM during treatment for PDA	9 (31%)	19 (56%)	0.029
Treatment for PDA	27(87%)	24(70%)	0.647
Feed type at reaching full feeds (150 ml/kg/day)
EBM	68 (88%)	64 (84%)	0.461
PDHM	18 (23%)	17 (22%)	0.882
PTF	1 (1.3%)	–	1.000

PIH-Pregnancy induced hypertension, APH – Ante-partum Haemorrhage, PPROM- Premature prolonged rupture of membrane, SGA – Small for Gestational age, CRIB Score-Clinical Risk Index for Babies score, PDA – Patent ductus arteriosus, EBM – Express breast milk, PDHM – Pasteurized human donor milk, PTF – Preterm formula, CLD- Chronic lung disease. Data summaries represent median (IQR, R) or N (%), as appropriate.

Mixed logistic regression analysis with a random intercepts model was performed to assess the effect of probiotic treatment on CRP levels from week 1 to week 13, while accounting for the correlation between weekly time points. Weeks 6 to 13 were combined to ensure sufficient numbers in the study group for the analysis. The model was adjusted for gestational age and median CRP over the first 3 days and an interaction effect between treatment and week was evaluated. The adjustment for CRP in the first three days of life was made to control for any differences in baseline health status of the infants between the treatments groups that were more likely associated with prenatal and immediate postnatal risk factors (e.g. chorioamnionitis, early onset sepsis). Results were summarized as adjusted odds ratios (OR) and accompanying 95% confidence intervals (CI). All tests were two-sided and a p-value <0.05 was considered statistically significant. SPSS (version 20.0, IBM SPSS) and SAS (version 9.3, Cary, NC, USA) statistical software were used for data analysis. P-values <0.05 were considered statistically significant.

3 Results

The baseline demographic characteristics of the infants in the RCT are shown in Table 1 and their clinical outcomes are shown in Table 2. There was no significant difference in baseline characteristics or clinical outcomes between the two treatment groups. Stool colonization with bifidobacteria was better in the probiotic group. Table 3 provides a weekly summary of CRP measurements for 76 probiotic and 75 placebo infants who had CRP’s taken. Overall, a median of 10 CRP measurements per infant (IQR: 5–20, R: 1–34) were taken in the probiotic versus 10 (IQR: 6–17, R: 2–38) the placebo group (p = 0.861) in the first 13 weeks after birth. On univariate analysis, there was no difference in the weekly median or peak CRP between the two treatment groups; the proportion of high CRP measurements was greater in week 5 in the probiotic group (15% vs 4% , p = 0.026), otherwise there was no difference between the two groups at any other weekly time point (Table 3).

Table 2
Clinical outcomes

Outcomes Probiotic (N = 77) Placebo (N = 76) p-value

All cause deaths – –

NEC ≥ Stage II – 1 0.497

Late onset sepsis 17(22%) 12(16%) 0.410

Time to reach full enteral feeds(150 ml/kg/d)in days 12(9–21; 5–71) 12(8–16; 3–81) 0.306

Length of hospital stay (w) 10(6–14; 2–61) 10(7–14; 3–60) 0.812

Discharge weight (g) 2590(2184–2990; 1565–4290) 2565(2303–3080; 1605–5074) 0.539

Early onset sepsis

Suspected 77(100%) 74(98%) 0.245

Proven 4(5%) 2(3%) 0.681

Duration of antibiotics(d) 3(3–5; 2–14) 3(3–5; 3–18) 0.685

Late onset sepsis

Suspected episode

None 48(62%) 43(57%) 0.744

1 15(20%) 16(21%)

2+ 14(18%) 17(22%)

Proven episode

None 60(78%) 64(84%) 0.465

1 12(16%) 10(13%)

2+ 5(7%) 2(3%)

Duration of antibiotics (d) 7(5–10; 3–21) 6(3–11; 2–33) 0.296

Outcomes	Probiotic (N = 77)	Placebo (N = 76)	p-value
All cause deaths	–	–
NEC ≥ Stage II	–	1	0.497
Late onset sepsis	17(22%)	12(16%)	0.410
Time to reach full enteral feeds(150 ml/kg/d)in days	12(9–21; 5–71)	12(8–16; 3–81)	0.306
Length of hospital stay (w)	10(6–14; 2–61)	10(7–14; 3–60)	0.812
Discharge weight (g)	2590(2184–2990; 1565–4290)	2565(2303–3080; 1605–5074)	0.539
Early onset sepsis
Suspected	77(100%)	74(98%)	0.245
Proven	4(5%)	2(3%)	0.681
Duration of antibiotics(d)	3(3–5; 2–14)	3(3–5; 3–18)	0.685
Late onset sepsis
Suspected episode
None	48(62%)	43(57%)	0.744
1	15(20%)	16(21%)
2+	14(18%)	17(22%)
Proven episode
None	60(78%)	64(84%)	0.465
1	12(16%)	10(13%)
2+	5(7%)	2(3%)
Duration of antibiotics (d)	7(5–10; 3–21)	6(3–11; 2–33)	0.296

Data summaries represent median (IQR, R) or N (%), as appropriate.

Table 3

CRP weekly summaries

	Placebo	Probiotics	p-value
Week 1	N = 75	N = 76
Median CRP	5 (5–8, 5–55)	5 (5–7.9, 5–22)	0.602
Peak CRP	9 (5–214, 5–187)	7.5 (5–15, 5–76)	0.452
CRP high (≥15 mg/L)	25 (33%)	22 (29%)	0.561
No. of CRP’s per baby	5 (4–7, 1–7)	5 (4–6, 1–7)	0.341
Week 2	N = 75	N = 76
Median CRP	5 (5–7, 5–42) (n = 46)	15 (5–8, 5–57) (n = 48)	0.500
Peak CRP	5 (5–10, 5–102.5) (n = 46)	5 (5–19, 5–119) (n = 48)	0.594
CRP high (>10 mg/L)	9 (12%)	16 (21%)	0.135
No. of CRP’s per baby	1 (0–3, 0–7)	1.5 (0–4, 0–7)	0.717
Week 3	N = 74	N = 74
Median CRP	5 (5–7, 5–44) (n = 42)	5 (5–6, 5–54) (n = 39)	0.453
Peak CRP	5 (5–14, 5–58) (n = 42)	5 (5–15, 5–119) (n = 39)	0.520
CRP high (>10 mg/L)	14 (19%)	11 (15%)	0.519
No. of CRP’s per baby	1 (0–2, 0–7)	1 (0–3, 0–7)	0.901
Week 4	N = 73	N = 74
Median CRP	5 (5–7, 5–24) (n = 25)	5 (5–8, 5–43.5) (n = 32)	0.633
Peak CRP	5 (5–10, 5–44) (n = 25)	6 (5–24, 5–229) (n = 32)	0.265
CRP high (>10 mg/L)	6 (8%)	13 (18%)	0.085
No. of CRP’s per baby	0 (0–1, 0–7)	0 (0–2, 0–7)	0.187
Week 5	N = 68	N = 68
Median CRP	5 (5–6, 5–28) (n = 23)	5 (5–12, 5–126.5) (n = 26)	0.150
Peak CRP	5 (5–8, 5–80) (n = 23)	5.5 (5–22, 5–147) (n = 26)	0.072
CRP high (>10 mg/L)	3 (4%)	11 (15%)	0.026
No. of CRP’s per baby	0 (0–1, 0–7)	0 (0–1, 0–7)	0.459
Week 6+	N = 63	N = 62
Median CRP	5 (5–9, 4–17) (n = 35)	5 (5–5, 5–12) (n = 33)	0.085
Peak CRP	5 (5–22, 4–66) (n = 35)	5 (5–12, 5–42) (n = 33)	0.710
CRP high (>10 mg/L)	10 (13%)	8 (11%)	0.585
No. of CRP’s per baby	0 (0–2, 0–15)	0 (0–2.8, 0–13)	0.861

Data represents median, interquartile range, or N (%), as appropriate.

Table 4

Model summary showing odds ratios and 95% confidence intervals for ‘high’ vs ‘normal’ CRP with adjustment for gestational age and the median of CRP measurements in first 3 days after birth

High CRP level	Adjusted odds ratio	95% confidence interval	p-value
Group
Placebo	1.00
Probiotic	1.62	0.91–2.88	0.102
Week
1	1.00
2	0.37	0.20–0.68	0.001
3	0.35	0.19–0.65	0.001
4	0.26	0.13–0.49	<0.001
5	0.16	0.08–0.33	<0.001
6–13	0.21	0.11–0.41	<0.001
Gestational age^*
Median CRP in first 3 days^*

^*Adjusted for these variables.

In the adjusted analysis, there was no evidence of a difference between probiotic and placebo groups in reducing the proportion of high CRP levels over time (treatment by weekly time points interaction, p = 0.187), and no evidence of a difference in the proportion of high CRP measurements across all time points between probiotic and placebo groups (adjusted OR 1.62, 95% CI 0.91–2.88, p = 0.102) (Table 4). The proportion of high CRP measurements, regardless of treatment group, was significantly lower in each successive week compared to week 1 (Table 4). The proportion of infants with detectable fecal B. breve increased significantly after supplementation in the probiotic vs. control group (p < 0.001). NEC≥ Stage II occurred in only 1 infant (placebo group). There were no adverse effects including probiotic sepsis.

4 Discussion

This exploratory analysis of results from our previously reported RCT showed that there was no significant difference in the number of CRPs and the median CRP levels in preterm infants receiving probiotics or placebo. There was also no significant difference in the proportion of infants with high CRP levels at any stage during hospital stay.

Meta analyses of RCTs have shown that probiotics decrease the risk of NEC and LOS in preterm infants [11 –13]. Hence, theoretically it is possible CRP levels would be lower in the probiotic group. The possible reasons for not finding any significant differences in our RCT need to be discussed.

Due to the high chances of cross colonization, infants in the placebo group are known to get colonized with the probiotic organism. The recently published PIPS trial that randomized 1315 preterm infants to receive probiotic Bifidobacterium breve BBG-001 or placebo showed a very high cross colonisation rate of nearly 49% [21]. Similar findings have also been reported by Kitajima et al. [22]. It is possible that cross colonization might have occurred in our study, leading to the lack of differences in CRP levels between the two groups. We were not able to study the issue of cross colonization in our trial as the strain-specific probe did not work. The other possible explanation is the very low incidence of NEC in our study infants. Only one infant in the placebo group developed ≥ stage 2 NEC. Even though the incidence of LOS was relatively high in our study infants (19% in the probiotic group and 16% in the placebo group), it is likely that the CRP levels were not different because the incidence of LOS was similar both groups.

In a newborn animal model, Yoshikazu Ohtsuka et al. have shown that administration of B. breve m-16v resulted in significant reduction in the expression of inflammation-related genes, including lipoprotein lipase (Lpl), glutathione peroxidase 2 (Gpx2), and lipopolysaccharide-binding protein (Lbp), in the colon [23]. It is possible that the anti-inflammatory mechanisms of B. breve m16-v may be limited to the gut and hence CRP levels were not significantly different between the groups.

Studies in adults have shown that supplementation with probiotics decrease high sensitive CRP (hs-CRP) levels in diabetes [24] and depression [25]. In an observational study of patients with HIV on anti-retroviral therapy, probiotic supplementation resulted in significant reduction in hs-CRP levels, but not the regular CRP [26]. Since our lab measures conventional CRP (not hs-CRP), the true effects of probiotics may have been missed.

The strengths and limitations of our study need to be discussed. To our knowledge this is the first study reporting on CRP levels after probiotic vs. placebo supplementation in preterm infants. The validity of its results is high considering the data originates from a well-designed and rigorously conducted RCT. We therefore believe that the study makes a unique contribution in the field of probiotics for preterm infants. The limitations include the fact that standardized prospective monitoring of CRP was not part of the trial protocol. The design also makes it difficult to rule out the influence of confounders such as antibiotics, anti-inflammatory (e.g. indomethacin or ibuprofen), and postnatal exposure to steroid. Despite best efforts results of a post-hoc analysis can be used only for hypothesis generation.

In summary probiotic supplementation with B. breve m-16v did not decrease the CRP levels in preterm infants born <33 weeks gestation. Similar analyses from the recently conducted multicentre RCTs will provide further evidence on this subject. Future RCTs should consider using standardized prospective monitoring with high sensitivity CRP in addition to the conventional CRP measurements.

Footnotes

Acknowledgments

Nil

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